Anti tenascin c antibodies to treat inflammatory diseases

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DESCRIPTION

Biological materials and uses thereof

The present invention relates to tenascin C and its activity in chronic inflammation. Tenascin C modulators and their biological activity are also provided.

Inflammation is the complex biological response of tissues to harmful stimuli, such as pathogens, tissue damage or irritants. A protective attempt of the tissue is to remove the harmful stimuli as well as start the healing process for the tissue. The abnormalities associated with inflammation comprise a large unrelated group of disorders that underlie various human diseases (inflammatory disorders). Examples of diseases with an inflammatory appearance include (but not limited to) asthma, autoimmune disease, glomerulonephritis, allergy (hypersensitivity), inflammatory bowel diseases, reperfusion injury, rheumatoid arthritis and transplant rejection.

In particular, chronic inflammation is a debilitating and serious condition associated with many of the above diseases and is characterized by persistent inflammation at an infection or injury site, or in relation to altered immune responses such as in autoimmune disease.

Rheumatoid arthritis (RA) is a typical, but not the only, example of a chronic inflammatory condition. RA is characterized by synovial inflammation and destruction of cartilage and bone of the joints mediated by persistent synthesis of proinflammatory cytokines and matrix metalloproteinases (MMP). Biological compounds that suppress the synthesis of inflammatory cytokines such as TNF and IL-6 are successful in the treatment of short-term RA. However, repeated treatments are required, which makes it an expensive therapeutic approach, and does not provide long-term remission. In addition, total systemic suppression of cytokine function has inherent problems such as increased infectious risk. Therefore, despite advances in care, there remains an unmet need for an economical mode of chronic inflammation treatment that is effective in the long term (Smolen (2006) and Williams (2007)).

The mechanisms that support the chronicity of the disease remain unclear and the factor or factors that lead to prolonged expression of inflammatory and destructive mediators are currently unknown.

Toll-like receptors (TLRs) play a key role in driving the production of inflammatory mediators in RA and blocking TLR function can have significant clinical benefits (reviewed in Brentano (2005) and O'Neill (2002) ). This family of receptors forms an integral part of the immune system. TLRs mediate host defense against infection and injury by recognizing both pathogen-associated molecular patterns (PAMP) and damage-associated molecular patterns (DAMP) (Matzinger (2002)). DAMPs are endogenous proinflammatory molecules generated after tissue injury and include intracellular molecules released from damaged or necrotic cells, extracellular matrix fragment molecules (ECM) or positive regulated ECM molecules after injury (reviewed in Bianchi (2007) and Gordon ( 2002)).

After activation, TLRs promote both innate and adaptive immune responses including stimulation of proinflammatory cytokine and MMP expression (Medzhitov (2002)). TLRs are expressed at high levels in synovial tissue of patients with RA (Radstake (2004), Roelofs (2005), Sacre (2007), and Sacre, manuscript submitted in 2008) and mice are protected with targeted deletions or loss of function mutations in TLR4 of experimental arthritis (Choe (2003) and Lee (2005)). In addition, TLR4 inhibitors can reduce destructive arthritis in mice (Abdollahi-Roodsaz (2007)) and a potential TLR4 inhibitor improved symptoms in 15 of 23 patients with moderate to severe RA in a preliminary phase I trial (Vanags (2006)). However, it is not clear that the TLR ligand or ligands are involved in the pathogenesis of disease.

Tenascin C is an ECM glycoprotein that is associated with tissue injury and wound repair. Tenascin C is specifically expressed during active tissue remodeling during embryogenesis, being observed first during gastrulation and somite formation. In later stages of development the expression is restricted to sites of branching morphogenesis of the mammary gland and lung, in the developing skeleton, the cardiovascular system and in connective tissues at sites of transformation of epithelium to mesenchyme. Expression is negatively regulated once these processes cease and before embryogenesis is completed (Jones (2000)).

Tenascin C is not normally expressed in healthy adult tissue but, in adults, it is positively regulated specifically and transiently during acute inflammation and is persistently expressed in chronic inflammation (reviewed in Chiquet-Ehrismann (2003)). Immunohistochemical studies show that little tenascin C is expressed in normal human joints but levels greatly increase in synoviums of RA, in areas of inflammation and fibrosis, specifically below the synovial lining, in the invading pannus and around blood vessels. (Cutolo (1992), MacCachren (1992) and Salter (1993)). There is also a significant increase in tenascin C levels in synovial fluid of patients with RA (Chevalier (1994) and Hasegawa (2007)) and in cartilage with RA (Salter (1993) and Chevalier (1994)).

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Tenascin C is a large hexamerican protein of 1.5 million Da. Each chain comprises different domains, including an assembly domain (TA), EGF type repetitions (EGF-L), fibronectin type III (TNIII) repetitions and a fibrinogen (FBG) type balloon (reviewed in Orend (2005) ). Tenascin C sequences and their domains are shown in Figure 13.

Previously, the role of tenascin C in inflammation has been doubtful, with tests showing various effects on different immune cells. For example, tenascin C has been shown to support adhesion and lymphocyte bearing of primary human tonsils and peripheral blood, thereby suggesting a role in stimulating lymphocyte migration (Clark (1997)). In addition, mice without tenascin C show reduced lymphocyte infiltration and lower levels of IFN, TNF and IL-4 mRNA after concanavalin A-induced liver injury in mice (El-Karef (2007)). Therefore, evidence suggests that tenascin C is involved in promoting the activity of acute inflammatory cells. However, it has also been indicated that tenascin C inhibits monocyte chemotaxis in vitro (Loike (2001)) and mice without tenascin C show increased migration of monocytes and macrophages in breast tumor stroma (Talts (1999)). These tests therefore suggest that tenascin C has a role in the inhibition of inflammatory cells.

The inventors have shown that tenascin C is an endogenous TLR4 ligand that is required for the destructive joint inflammation seen in arthritis.

In addition, it has now been shown that tenascin C is not involved in the induction of inflammation (acute inflammatory response) but instead is involved in prolonging the inflammatory response that characterizes the chronic inflammatory condition. In particular, it has now been shown that tenascin C is an endogenous activator of TLR4 and it has been shown that this molecule is required for destructive joint inflammation.

A role of tenascin C has been demonstrated in the mediation of an immune response in the joints by induction of joint inflammation after intra-articular injection of the FBG domain of tenascin C in mice in vivo. In addition, acute inflammation of the joints induced by zymosan was not as prolonged in mice deficient in tenascin C. Both wild-type and non-tenascin C mice responded to induction of acute inflammation by zymosan alike, demonstrating that tenascin C does not appear to be involved in the onset of inflammation. However, the less persistent synovitis shown by mice without tenascin C indicates a role in maintaining joint inflammation. The importance of tenascin C in prolonging joint inflammation was underscored by the observation that the targeted deletion of tenascin C protected mice from sustained erosive joint inflammation during arthritis induced by mBSA immunization.

It has now been shown that tenascin C is capable of activating cells in the joint and the primary active domain of tenascin C has been mapped into the fibrinogen-type globule (FBG), a globular domain of 227 amino acids (26.9 kDa) at the C-terminal end of the molecule (Siri (1991)).

The addition of FBG to synovial membrane cultures of patients with RA enhanced the spontaneous release of proinflammatory cytokines. It also stimulated the synthesis of TNF, IL-6 and IL-8 in primary human macrophages and IL6 in synovial fibroblasts of AR by activating signaling pathways dependent on TLR4 and MyD88.

It has now been shown that, as in the case of LPS, the expression of TLR4 is necessary for the induction of cytokine synthesis by FBG. However, unlike LPS, neither CD14 nor MD-2 seems to be required for TLR-4 activation. CD14 is dispensable for the activation of TLR4 by other ligands. TLR4 is not required to respond to lipid A in a MyD88-dependent manner (Jiang (2005)), EDA fibronectin can activate mast cells even in the absence of CD14 (Gondokaryono (2007)) and THP-1 cell hyaluronic acid activation Human monocytes require a complex of TLR4, CD44 and MD-2, but not CD14 (Taylor (2007)).

The formation of receptor complexes defined by each TLR4 ligand can facilitate the recruitment of different intracellular signaling molecules / adapters. This may explain the differential cellular responses observed with FBG and LPS, for example lack of induction of IL-8 by FBG in synovial fibroblasts with RA. Similarly, activation by hyaluronic acid of the TLR4 and CD44 complex induces a pattern of gene expression in mouse alveolar macrophage cell lines that is different from LPS (Taylor (2007)). That FBG induces synthesis of IL-8 in human macrophages suggests that recognition and / or signaling of specific cell-type ligand occurs.

The tightly regulated pattern of tenascin C expression makes it an attractive target for the treatment of chronic inflammation. It is predominantly absent from healthy adults, however expression is specifically induced after tissue injury. During acute inflammation, tenascin C is expressed transiently: induction often precedes inflammation and both mRNA and tissue protein are absent when the inflammation has resolved (reviewed in Chiquet-Ehrismann (2003)).

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It has now been shown that persistent expression of tenascin C is associated with chronic inflammation. In addition to RA, increased levels of tenascin C have been observed in other autoimmune diseases including multiple sclerosis (Gutowski (1999)) and Sjogren's disease (Amin (2001)), and in non-healing wounds and diabetic and venous ulcers (Loots (1998)). The de novo synthesis of tenascin C correlates well with the intensity of inflammation in diseases of the oral mucosa and plasma levels of tenascin C are a reliable indicator of the activity of inflammatory bowel diseases before and after medication or surgery ( reviewed in Chiquet-Ehrismann (2003)).

In a first aspect of the invention there is provided an agent for the modulation of a chronic inflammatory response in which the agent modulates the biological activity of tenascin C, the agent being an antibody or antigen-binding fragment thereof having specificity for the FBG domain of tenascin C.

The agent of the first aspect of the invention can modulate the biological activity of tenascin C by altering the binding properties of tenascin C.

Also described herein are agents that can modulate the biological activity of tenascin C by altering the transcription and / or translation of tenascin C.

Such agents can be identified using methods well known in the art, such as:

<dl><dt>(to) </dt><dd>determining the effect of a test agent on the levels of tenascin C expression, for example by Southern blotting or related hybridization techniques; </dd></dl>

<dl><dt>(b) </dt><dd>determining the effect of a test agent on tenascin C protein levels, for example by immunoassays using anti tenascin C antibodies; and</dd></dl>

<dl><dt>(c) </dt><dd>determining the effect of a test agent on a functional marker or result of tenascin C activity, for example by the methods of the examples. </dd></dl>

The agents disclosed herein can negatively regulate the biological activity of tenascin

C.

The agents disclosed herein can positively regulate the biological activity of tenascin

C. The adequacy of positive regulation of the activity of immune and inflammatory molecules and cells is relevant for the production of therapies for immunocompromised and inflammatory patients and in the development of vaccines (see Harandi (2009)).

The agent described herein may be a transcription inhibitor of tenascin C.

The agent described herein may be a translation inhibitor of tenascin C.

The agent of the first aspect of the invention may be an inhibitor of tenascin C binding properties. For example, the agent may alter the formation of tenascin C so that it is no longer capable of binding with its receptor.

The agent of the first aspect of the invention can be a competitive binding inhibitor of tenascin C. It will be appreciated by those skilled in the art that the agent can also inhibit the biological activity of tenascin C by blocking the function of the tenascin C receptor directly (acting as a tenascin C receptor antagonist) or indirectly (binding with intermediary molecules or assistants).

The agent of the first aspect of the invention may be a TLR-4 receptor antagonist.

It will be appreciated by those skilled in the art that the inhibition of the biological activity of tenascin C by an agent of the invention can be complete or partial. For example, the agent can inhibit the biological activity of tenascin C by at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, and more preferably 100% compared to the biological activity of tenascin C in inflammatory cells that have not been exposed to the agent.

The agent described herein can be selected from the group consisting of short interfering RNA molecules (siRNA), short hairpin RNA molecules (hRNA), antisense oligonucleotides, compounds with affinity for tenascin C binding, antibodies (polyclonal or monoclonal) and antigen binding fragments thereof, small inhibitor compounds, polypeptides and proteins.

In one example the agent is an siRNA. RNA interference is a two stage process. The first stage, which is called the start-up stage, is digested input RNAb in small interfering RNA (siRNA) of 21-23 nucleotides (nt), probably by the action of Dicer, a member of the Rnasa III family of specific rRNA ribonucleases, which processes (cleaves) cRNA (introduced directly or by a transgene or a virus) in an ATP-dependent manner. Successive cleavage events degrade RNA to double strands

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19-21 bp (siRNA) each with 3 'protrusions of 2 nucleotides (Hutvagner and Zamore, 2002, Curr. Opin. Genetics and Development 12: 225-232; Bernstein, 2001, Nature 409: 363-366).

In the effector stage, the double siRNA chains bind with a nuclease complex to form the RNA-induced muffler complex (RISC). An ATP-dependent unwinding of the double strand of siRNA is required for RISC activation. The active RISC is then directed to the homologous transcript by base pair formation interactions and cleaves the mRNA into 12 nucleotide fragments from the 3 'terminal end of the siRNA (Hutvagner and Zamore, 2002, mentioned above .; Hammond et al., 2001 , Nat. Rev. Gen. 2: 110-119 (2001); Sharp, 2001, Genes. Dev. 15: 485-90). Although the cleavage mechanism has yet to be elucidated, research indicates that each RISC contains a single siRNA and an RNase (Hutvagner and Zamore, 2002, mentioned above.).

In view of the remarkable potency of RNAi, an amplification stage within the RNAi pathway has been suggested. Amplification could occur by copying the input rRNAs which would generate more siRNAs, or by replication of the formed siRNAs. Alternatively, or additionally, the amplification could be performed by multiple RISC renewal events (Hammond et al., 2001, mentioned above; Hutvagner and Zamore, 2002, mentioned above.). Additional information on RNAi can be found in the following reviews, Tuschl, 2001, Chem. Biochem. 2: 239-245, Cullen, 2002, Nat. Immunol. 3: 597-599 and Brantl, 2002, Biochem. Biophys Act. 1575: 15-25.

The synthesis of RNAi molecules suitable for use with the present invention can be carried out as follows. First, the tenascin C mRNA sequence is explored downstream of the AUG start codon with respect to AA dinucleotide sequences. The occurrence of each AA and the 19 adjacent 3 'nucleotides is recorded as potential siRNA target sites. Preferably, the siRNA target sites are selected from the open reading phase, since the untranslated regions (UTR) are richer at regulatory protein binding sites. UTR-binding proteins and / or translation initiation complexes can interfere with the binding of the siRNA endonuclease complex (Tuschl, ChemBiochem. 2: 239-245). It will be appreciated, however, that siRNAs targeting untranslated regions can also be effective.

Second, potential target sites are compared with an appropriate genomic database (eg, human, mouse, rat, etc.) using sequence alignment software, such as BLAST (www.ncbi.nlm.nih .gov / BLAST /). Potential target sites showing significant homology with other coding sequences are filtered off.

Target sequences classified as template for siRNA synthesis are selected. Preferred sequences are those that include a low G / C content since these have been shown to be more effective in mediating gene silencing compared to those of G / C content greater than 55%. Several target sites are preferably selected along the length of the target gene for evaluation. For a better evaluation of the selected siRNAs, a negative control is preferably used together. The negative control siRNA preferably includes the same nucleotide composition as the siRNAs but lacks significant homology with the genome. Therefore, a mixed nucleotide sequence of the siRNA is preferably used, provided there is no significant homology with any other gene.

Suitable siRNA molecules can be synthesized as described above so that they are complementary and therefore bind to the complete nucleotide sequence of tenascin C or parts thereof. The nucleotide sequence of tenascin C is found in Figure 14.

In one example the agent can be a short hairpin RNA (hRNA).

A small hairpin RNA or short hairpin RNA (hRNA) is an RNA sequence that performs a narrow hairpin turn that can be used to silence gene expression by RNA interference. The hRNA uses a vector (usually adenovirus or lentivirus) introduced into cells and uses the U6 promoter to ensure that the hRNA is always expressed. This vector is usually passed to the descending cells, allowing gene silencing to be inherited. The hairpin structure of the hRNA is cleaved by the cellular machinery in siRNA, which is then attached to the RNA-induced muffler complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it binds. (McIntyre (2006) and Paddison (2002)).

The agent of the first aspect of the invention may be a tenascin C domain or variant thereof. It has been shown that the FBG domain is predominantly involved in the interaction of tenascin C with its target in relation to the persistence of chronic inflammation. Consequently, the preferred domain is the FBG domain (sequence shown in Figure 13) or variants thereof.

In an alternative example, the agent is an antisense oligonucleotide.

The design of antisense molecules that can be used to effectively reduce the levels / activity of tenascin C requires consideration of two important aspects for the antisense approach. The first aspect is the

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supply of the oligonucleotide in the cytoplasm of cancer cells, while the second aspect is the design of an oligonucleotide that specifically binds to the designated mRNA within cells such that it inhibits translation thereof.

The prior art teaches several delivery strategies that can be used to effectively deliver oligonucleotides to a wide variety of cell types (e.g., see Luft, 1998, J Mol Med 76: 75-6; Kronenwett et al., 1998, Blood 91: 852-62; Rajur et al., 1997, Bioconjug Chem 8: 935-40; Lavigne et al., 1997, Biochem Biophys Res Commun 237: 566-71; Aoki et al., 1997, Biochem Biophys Res Commun 231: 540 -5).

In addition, algorithms are available to identify the sequences with the highest binding affinity predicted by their target mRNA based on a thermodynamic cycle that explains the energy of structural alternations in both the target mRNA and the oligonucleotide (for example, see Walton et al. ., 1999, Biotechnol Bioeng 65: 1-9).

Several approaches to designing and predicting the efficacy of specific oligonucleotides using an in vitro system are also known (for example, see Matveeva et al., 1998, Nature biotechnology 16: 1374-1375).

Several clinical trials have demonstrated safety, feasibility and activity of antisense oligonucleotides. For example, antisense oligonucleotides suitable for cancer treatment have been used successfully (Holmlund et al., 1999, Curr Opin Mol Ther 1: 372-85; Gerwitz, 1999, Curr Opin Mol Ther 1: 297-306). More recently, it has been indicated that antisense-mediated suppression of human heparanase gene expression inhibits the pleural spread of human cancer cells in a mouse model (Uno et al., 2001, Cancer Res 61: 7855-60).

Therefore, those skilled in the art are easily able to design and implement appropriate antisense approaches to modulate the expression of tenascin C.

Advantageously, the antisense oligonucleotide is 15 to 35 bases in length. For example, it has been shown that oligonucleotides of 20 units inhibit mRNA expression of the epidermal growth factor receptor (Witters et al, Breast Cancer Res Treat 53: 41-50 (1999)) and it has been shown that oligonucleotides of 25 units reduce the expression of adrenocorticotropic hormone by more than 90% (Frankel et al, J Neurosurg 91: 261-7 (1999)). However, it is appreciated that it may be desirable to use oligonucleotides with lengths outside this range, for example 10, 11, 12, 13 or 14 bases, or 36, 37, 38, 39 or 40 bases.

It will be further appreciated by those skilled in the art that oligonucleotides are subject to degradation or inactivation by cellular endogenous nucleases. To counteract this problem, it is possible to use modified oligonucleotides, for example having altered internucleotide bonds, in which naturally occurring phosphodiester bonds have been replaced with another link. For example, Agrawal et al (1988) Proc. Natl Acad. Sci. USA 85, 7079-7083 showed increased inhibition in tissue culture of HIV-1 using phosphoramidate oligonucleotide and phosphorothioates. Sarin et al (1988) Proc. Natl Acad. Sci. USA 85, 7448-7451 demonstrated increased inhibition of HIV-1 using oligonucleotide methylphosphonates. Agrawal et al (1989) Proc. Natl Acad. Sci. USA 86, 7790-7794 showed inhibition of HIV-1 replication in both early infected and chronically infected cell cultures, using nucleotide sequence specific phosphorothioates oligonucleotide. Leither et al (1990) Proc. Natl Acad. Sci. USA 87, 3430-3434 indicate the inhibition in tissue culture of influenza virus replication by oligonucleotide phosphorothioates.

Oligonucleotides that have artificial bonds have been shown to be resistant to degradation in vivo. For example, Shaw et al (1991) in Nucleic Acids Res. 19, 747-750, indicate that oligonucleotides not otherwise modified become more resistant to nucleases in vivo when blocked at the 3 'end by certain protective structures and that unprotected oligonucleotide phosphorothioates do not degrade in vivo.

A detailed description of the H-phosphonate approach to the synthesis of oligonucleoside phosphorothioates is provided in Agrawal and Tang (1990) Tetrahedron Letters 31, 7541-7544, the teachings of which are hereby incorporated herein by reference. Synthesis of oligonucleoside methylphosphonates, phosphorodithioates, phosphoramidates, phosphate esters, linked phosphoramidates and linked phosphorothioates are known in the art. See, for example, Agrawal and Goodchild (1987) Tetrahedron Letters 28, 3539; Nielsen et al (1988) Tetrahedron Letters 29, 2911; Jager et al (1988) Biochemistry 27, 7237; Uznanski et al (1987) Tetrahedron Letters 28, 3401; Bannwarth (1988) Helv. Chim. Minutes 71, 1517; Crosstick and Vyle (1989) Tetrahedron Letters 30, 4693; Agrawal et al (1990) Proc. Natl Acad. Sci. USA 87, 1401-1405, whose teachings are incorporated herein by reference. Other methods for synthesis or production are also possible. In a preferred embodiment the oligonucleotide is a deoxyribonucleic acid (DNA), although ribonucleic acid (RNA) sequences can also be synthesized and applied.

The oligonucleotides useful in the examples described herein are preferably designed to resist degradation by endogenous nucleolytic enzymes. In vivo degradation of oligonucleotides produces oligonucleotide degradation products of reduced length. These degradation products are more likely to perform non-specific hybridization and are less likely to be effective in relation to their

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full length counterparts. Therefore, it is desirable to use oligonucleotides that are resistant to degradation in the body and that are capable of reaching the target cells. The present oligonucleotides can be made more resistant to degradation in vivo by replacing with one or more internal artificial internucleotide bonds the native phosphodiester bonds, for example, replacing phosphate with sulfur in the bond. Examples of linkages that may be used include phosphorothioates, methylphosphonates, sulfone, sulfate, cetyl, phosphorodithioates, various phosphoramidates, phosphate esters, linked phosphorothioates and linked phosphoramidates. Such examples are illustrative, rather than limiting, since other internucleotide bonds are well known in the art. The synthesis of oligonucleotides having one or more of these bonds replacing phosphodiester internucleotide bonds is well known in the art, including synthetic routes for producing oligonucleotides having mixed internucleotide bonds.

Oligonucleotides can be made resistant to spread by endogenous enzymes by "terminal coating"

or incorporating similar groups in the 5 'or 3' terminal nucleotides. A terminal coating reagent is commercially available as Amino-Link II ™ from Applied BioSystems Inc, Foster City, CA. Methods for terminal coating are described, for example, in Shaw et al (1991) Nucleic Acids Res. 19, 747-750 and Agrawal et al (1991) Proc. Natl Acad. Sci. USA 88 (17), 7595-7599.

An additional method to prepare nuclease attack resistant oligonucleotides is to "self-stabilize" them as described in Tang et al (1993) Nucl. Acids Res. 21, 2729-2735. Self-stabilized oligonucleotides have hairpin loop structures at their 3 'ends, and show increased resistance to phosphodiesterase degradation of snake venom, DNA polymerase I and fetal bovine serum. The self-stabilized region of the oligonucleotide does not interfere with hybridization with complementary nucleic acids, and studies of pharmacokinetics and stability in mice have shown increased in vivo persistence of self-stabilized oligonucleotides with respect to their linear counterparts.

In an example in which the agent is a compound with tenascin C binding affinity, the compound may be substantially reversibly or substantially irreversibly bound to an active tenascin C site. In a further example, the compound may be linked with a part of tenascin C which is not the active site to interfere with the binding of tenascin C with a ligand or receptor. In a further example, the compound can be linked with a part of tenascin C to reduce the activity of proteins by an allosteric effect. This allosteric effect may be an allosteric effect that is involved in the natural regulation of tenascin C activity, for example in the activation of tenascin C by an "upstream activator".

Methods for detecting interactions between a test compound and tenascin C are well known in the art. For example, ultrafiltration can be used with ion spray / HPLC mass spectroscopy methods or other physical and analytical methods. In addition, Fluorescent Energy Resonance Transfer (FRET) methods can be used, in which the binding of two fluorescently labeled entities can be measured by measuring the interaction of the fluorescent markers when they are in close proximity to each other.

Alternative methods of detecting the binding of a polypeptide with macromolecules, for example DNA, RNA, proteins and phospholipids, include a surface plasmon resonance assay, for example as described in Plant et al., 1995, Analyt Biochem 226 ( 2), 342-348. The methods may make use of a polypeptide that is labeled, for example with a radioactive or fluorescent label.

An additional method for identifying a compound that is capable of binding to the polypeptide is one in which the polypeptide is exposed to the compound and any binding of the compound with said polypeptide is detected and / or measured. The binding constant for binding of the compound to the polypeptide can be determined. Suitable methods for detecting and / or measuring (quantifying) the binding of a compound with a polypeptide are well known to those skilled in the art and can be performed, for example, using a method capable of operating at high yield, for example a method based in microplate A new technology, called VLSIPS ™, has allowed the production of extremely small microplates containing hundreds of thousands or more different molecular probes. These biological microplates or matrices have probes arranged in matrices, with each probe assigned to a specific location. Biological microplates have been produced in which each location has a scale of, for example, ten micrometers. The microplates can be used to determine if target molecules interact with any of the probes in the microplate. After exposing the matrix to target molecules under selected test conditions, scanning devices can examine each location in the matrix and determine if a target molecule has interacted with the probe at that location.

Another method for identifying compounds with tenascin C binding affinity is the two yeast hybrid system, in which the polypeptides of the invention can be used to "capture" proteins that bind with tenascin C. The two yeast hybrid system It is described in Fields and Song, Nature 340: 245-246 (1989).

In a further example, the agent is a compound that has a ligand binding capacity for tenascin C.

For example, the agent may be a soluble fragment of a tenascin C receptor (such as FPRL1). Alternatively, the agent can be a high affinity molecule that mimics an antibody (a so-called "affibody")

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(for example, see document US 5,831,012 and www.affibody.se). These ligands are small, simple proteins composed of a bundle of three helices based on the framework of one of the IgG binding domains of Protein A (a surface protein of the Staphylococcus aureus bacteria). This framework has excellent characteristics as an affinity ligand and can be designed to bind with high affinity with any given target protein.

The agent of the first aspect of the invention is an antibody or antigen binding fragment thereof. The antigen-binding fragment can be selected from the group consisting of Fv fragments (for example single-chain Fv and Fv with disulfide bonds), Fab-type fragments (for example Fab fragments, Fab 'fragments and F (ab) 2 fragments), domains individual variables (eg, VH and VL domains) and domain antibodies (dAb, including single and double formats [ie dAb-linker-dAb]).

The antibody may preferably bind specifically to the FBG domain that activates TLR4.

The advantages of using antibody fragments, instead of whole antibodies, are multiple. The smaller size of the fragments can lead to better pharmacological properties, such as better solid tissue penetration. In addition, antigen-binding fragments such as Fab, Fv, ScFv and dAb antibody fragments can be expressed in and secreted from E. coli, thereby allowing easy production of large amounts of said fragments.

Also included within the scope of the invention are modified versions of antibodies and an antigen binding fragment thereof, for example modified by covalent binding of polyethylene glycol or other suitable polymer.

Methods for generating antibodies and antibody fragments are well known in the art. For example, antibodies can be generated by any one of several methods that employ the induction of in vivo production of antibody molecules, immunoglobulin library screening (Orlandi. Et al, 1989. Proc. Natl. Acad. Sci. USA 86: 3833 -3837; Winter et al., 1991, Nature 349: 293-299) or generation of monoclonal antibody molecules by cultured cell lines. These include, but are not limited to, the hydridoma technique, the human B lymphocyte hybridoma technique, and the Epstein-Barr virus (EBV) hybridoma technique (Kohler et al., 1975. Nature 256: 4950497; Kozbor et al., 1985. J. Immunol. Methods 81: 31-42; Cote et al., 1983. Proc. Natl. Acad. Sci. USA 80: 2026-2030; Cole et al., 1984. Mol. Cell. Biol 62: 109-120).

Suitable monoclonal antibodies can be prepared for antigens selected by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications" JGR Hurrell (CRC Press, 1982).

Antibody fragments can be obtained using methods well known in the art (see, for example, Harlow and Lane, 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, New York). For example, antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (for example culture of Chinese hamster ovary cells or other protein expression systems) of DNA encoding the fragment. Alternatively, antibody fragments can be obtained by digestion with pepsin or papain of whole antibodies by conventional methods.

It will be appreciated by those skilled in the art that for human therapy or diagnosis, humanized antibodies are preferably used. Humanized forms of non-human antibodies (eg, murine) are genetically engineered chimeric antibodies or antibody fragments that preferably have minimal parts derived from non-human antibodies. Humanized antibodies include antibodies in which complementarity determining regions of a human antibody (receptor antibody) are replaced by residues of a complementarity determining region of a non-human species (donor antibody) such as mouse, rat or rabbit having the functionality desired. In some cases, conserved Fv framework moieties of the human antibody are replaced by corresponding non-human moieties. Humanized antibodies may also comprise residues that are neither found in the recipient antibody nor in the conserved framework sequences or in the complementary complementarity determining region. In general, the humanized antibody will comprise substantially all of at least one, and usually two, variable domains, in which all or substantially all complementarity determining regions correspond to those of a non-human antibody and all, or substantially all, regions Preserved framework correspond to those of a relevant human consensus sequence. Humanized antibodies also optimally include at least a portion of a constant antibody region, such as an Fc region, normally derived from a human antibody (see, for example, Jones et al., 1986. Nature 321: 522-525; Riechmann et al., 1988, Nature 332: 323-329; Presta, 1992, Curr. Op. Struct. Biol. 2: 593-596).

Methods for humanizing non-human antibodies are well known in the art. In general, the humanized antibody has one or more amino acid residues introduced into it from a source that is not human. These non-human amino acid residues, often referred to as imported residues, are usually taken from an imported variable domain. Humanization can be carried out essentially as described (see, for example,

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Jones et al., 1988, Nature 321: 522-525; Reichmann et al., 1988. Nature 332: 323-327; Verhoeyen et al., 1988, Science 239: 1534-1536I; US 4,816,567) replacing human complementarity determining regions with corresponding rodent complementarity determining regions. Consequently, said humanized antibodies are chimeric antibodies, in which substantially less than one domain

5 intact human variable has been replaced by the corresponding sequence of a non-human species. In practice, humanized antibodies can normally be human antibodies in which some complementarity determining region moieties and possibly some framework moieties conserved by rodent antibody analog sites are substituted.

10 Human antibodies can also be identified using various techniques known in this field, including phage display libraries (see, for example, Hoogenboom and Winter, 1991, J. Mol. Biol. 227: 381; Marks et al., 1991, J. Mol. Biol. 222: 581; Cole et al., 1985, In: Monoclonal antibodies and Cancer Therapy, Alan R. Liss, pp. 77; Boerner et al., 1991. J. Immunol. 147: 86-95).

fifteen Once suitable antibodies have been obtained, they can be tested for activity, for example by ELISA.

The agent of the first aspect of the invention may be an antibody or antigen-binding fragment thereof having specificity for the Toll Type 4 Receptor (TLR4), Toll Type 4 Receptor correceptors (at junction 20 of tenascin-4 , tenascin-C or a domain thereof of any of these).

Correceptors for primary receptors, such as TLR4, aid in the binding of a signaling molecule to the primary receptor to facilitate recognition and ligand binding and initiate / maintain the biological process resulting from receptor binding.

25 The agent of the first aspect of the invention may be an antibody or antigen-binding fragment thereof that has specificity for the tenascin C FBG domain.

Also described herein is a method for identifying an agent that modulates the activity of tenascin C comprising the steps of:

<dl><dt>(i)</dt><dd> provide one or more candidate agents; </dd></dl>

<dl><dt>(ii)</dt><dd> contacting one or more cells with tenascin C and the candidate agent (s); </dd></dl>

(iii) contact one or more cells with tenascin C and no candidate agent;

35 (iv) determine whether said candidate agent modulates the effect of tenascin C on the cell (s) in step (ii) compared to the cell or cells of the control stage (iii).

Methods can be carried out to determine if the candidate agent modulates the effect of tenascin C using the methods of the examples. 40 The method may result in a positive regulation of tenascin C activity.

The method may result in tenascin C activity being negatively regulated.

Four. Five The method may include the cells of steps (ii) and (iii) (described above) that express Toll 4 type receptor (TLR4).

The method may have the cell (s) selected from the group consisting of inflammatory cells, fibroblasts, fibroblast-like cells (including synovial fibroblasts of AR, also known as synoviocytes), 50 mouse embryonic fibroblasts, human embryonic kidney cells.

Inflammatory cells can be selected from the group consisting of macrophages, dendritic cells, monocytes, lymphocytes, monocyte cells and macrophage cells.

55 Also described herein is a method of identifying an agent that modulates a chronic inflammatory response by performing the method of identifying an agent that modulates the activity of tenascin C.

In this method chronic inflammation can be associated with any condition associated with inappropriate inflammation. Such conditions include, but are not limited to, rheumatoid arthritis (RA), autoimmune conditions, diseases.

60 inflammatory bowel, wounds that do not heal, multiple sclerosis, cancer, atherosclerosis, sjogren's disease, diabetes, lupus erythematosus (including systemic lupus erythematosus), asthma, fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UV damage and psoriasis .

It is of particular, but not exclusive, interest that chronic inflammation is associated with rheumatoid arthritis (RA). 65

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An agent identified in accordance with the method of the second and third aspects of the invention is also described herein. Said agent can modulate a chronic inflammatory response.

The agent can negatively regulate the chronic inflammatory response.

The agent can positively regulate the chronic inflammatory response.

The agent can be selected from the group consisting of short-interfering RNA (siRNA) molecules, short-hair RNA (hRNA) molecules, antisense oligonucleotides, compounds with affinity for tenascin C binding, antibodies (polyclonal or monoclonal) and fragments of antigen binding thereof, small inhibitor compounds, polypeptides and proteins.

In the first aspect of the invention chronic inflammation can be associated with any condition associated with inappropriate inflammation. Such conditions include, but are not limited to, rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel diseases, wounds that are not cured, multiple sclerosis, cancer, atherosclerosis, sjogren's disease, diabetes, lupus erythematosus (including systemic lupus erythematosus), asthma, fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UV damage and psoriasis.

In a second aspect of the invention there is provided a composition comprising an agent as defined in the first aspect of the invention and a pharmaceutically acceptable carrier, excipient and / or diluent.

It will be appreciated by those skilled in the art that said effective amount of the agent or formulation thereof can be supplied as a single-dose dose (ie acute administration) or, more preferably, as a series of doses over time (it is say chronic administration).

The agents of the invention can be formulated at various concentrations, depending on the efficacy / toxicity of the compound used and the indication for which it is used. Preferably, the formulation comprises the agent of the invention at a concentration of between 0.1 M and 1 mM, more preferably between 1 M and 100 M, between 5 M and 50 M, between 10 M and 50 M, between 20 M and 40 M and more preferably about 30 M. For in vitro applications, the formulations may comprise a lower concentration of a compound of the invention, for example between 0.0025 µM and 1 µM.

It will be appreciated by those skilled in the art that the agents of the invention will generally be administered in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with respect to the intended route of administration and conventional pharmaceutical practice (for example, see Remington: The Science and Practice of Pharmacy, 19th edition, 1995, Ed. Alfonso Gennaro, Mack Publishing Company, Pennsylvania; United States).

For example, the agents of the invention can be administered orally, orally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavoring or coloring agents, for immediate release applications, delayed or controlled. The agents of the invention can also be administered by intracavernous injection.

Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn starch, potato or tapioca), sodium starch glycolate, croscarmellose sodium and certain complex silicates , and granulation binders such as polyvinylpyrrolidone, hydroxypropyl methylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and gum arabic. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type can also be used as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and / or elixirs, the compounds of the invention may be combined with various sweetening or flavoring agents, coloring matter or dyes, with emulsifying and / or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

The agents of the invention can also be administered parenterally, for example, intravenously, intraarticularly, intraarterially, intraperitoneally, intrathecally, intraventricularly, intratrasternally, intracranially, intramuscularly. or subcutaneously, or they can be administered by infusion techniques. They are best used in the form of a sterile aqueous solution that may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. Aqueous solutions should be adequately buffered (preferably at a pH of 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is easily achieved by conventional pharmaceutical techniques well known to those skilled in the art.

Formulations suitable for parenteral administration include sterile aqueous and non-aqueous injection solutions that may contain antioxidants, buffers, bacteriostatics and solutes that make the formulation

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isotonic with the intended recipient's blood; and sterile aqueous and non-aqueous suspensions that may include suspending agents and thickening agents. The formulations can be presented in unit dose or multi-dose containers, for example sealed ampoules and bottles, and can be stored in a cryo-dried (lyophilized) condition that requires only the addition of the sterile liquid vehicle, for example water for injections, immediately before use. . Solutions and suspensions for extemporaneous injection of sterile powders, granules and tablets of the type previously described can also be prepared.

For oral and parenteral administration to human patients, the daily dosage level of the agents of the invention will usually be 1 to 1000 mg per adult (i.e. approximately 0.015 to 15 mg / kg) administered in individual or divided doses.

The agents of the invention can also be administered intranasally or by inhalation and are conveniently supplied in the form of a dry powder inhaler or an aerosol spray presentation of a pressurized container, pump, sprayer or nebulizer with the use of a suitable propellant. , for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A3) or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a measured amount. The pressurized container, pump, sprayer or nebulizer may contain a solution or suspension of the active compound, for example using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, for example, sorbitan trioleate. Capsules and cartridges (made, for example, of gelatin) may be formulated for use in an inhaler or insufflator to contain a powder mixture of a compound of the invention and a suitable powder base such as lactose or starch.

Aerosol or dry powder formulations are preferably arranged so that each measured dose or "discharge" contains at least 1 mg of a compound of the invention for delivery to the patient. It will be appreciated that the overall daily dose with an aerosol will vary from patient to patient, and can be administered in a single dose or, more commonly, in divided doses throughout the day.

Alternatively, the agents of the invention can be administered in the form of a suppository or pessary, or they can be applied topically in the form of a lotion, solution, cream, ointment or powder for external use. The compounds of the invention can also be administered transdermally, for example, by the use of a skin patch. They can also be administered by the eye.

For ophthalmic use, the agents of the invention may be formulated as micronized suspensions in isotonic saline solution, of adjusted pH, sterile or, preferably, as solutions in isotonic saline solution, of adjusted pH, sterile, optionally in combination with a preservative such as chloride Benzalkonium Alternatively, they can be formulated in an ointment such as petroleum jelly.

For topical application to the skin, the agents of the invention can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, petrolatum white, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Formulations suitable for topical administration in the mouth include dragees comprising the active ingredient in a flavored base, usually sucrose and gum arabic or tragacanth; pills comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and gum arabic; and mouthwashes comprising the active substance in a suitable liquid vehicle.

When the agent is a polypeptide, it may be preferable to use an extended release drug delivery system, such as microspheres. These are specifically designed to reduce the frequency of injections. An example of such a system is the Nutropin Deposit that encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that, once injected, release rhGH slowly over a prolonged period.

Alternatively, polypeptide agents of the present invention can be administered by a surgically implanted device that releases the drug directly to the required site.

Electroporation therapy (EPT) systems can also be used for the administration of proteins and polypeptides. A device that supplies an electric field by pulses to cells increases the permeability of cell membranes to the drug, resulting in a significant potentiation of the intracellular pharmacological supply.

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Proteins and polypeptides can also be supplied by electroincorporation (EI). IE occurs when small particles of up to 30 micrometers in diameter on the surface of the skin undergo electrical pulses identical or similar to those used in electroporation. In IE, these particles are conducted through the stratum corneum and into the deeper layers of the skin. The particles can be loaded or coated with drugs or genes or can simply act as "bullets" that generate pores in the skin through which drugs can enter.

An alternative method of supplying proteins and polypeptides is the thermosensitive ReGel injection. Below body temperature, ReGel is an injectable liquid while at body temperature it immediately forms a gel reservoir that slowly degrades and dissolves in known, safe, biodegradable polymers. The active drug is supplied over time as the biopolymers dissolve.

Protein and polypeptide pharmaceuticals can also be supplied orally. One such system employs a natural process for the oral uptake of vitamin B12 in the body to jointly deliver proteins and polypeptides. By coupling to the vitamin B12 uptake system, the protein or polypeptide can move through the intestinal wall. Complexes occur between analogs of vitamin B12 and the drug that retain both significant affinity for the intrinsic factor (FI) in the vitamin B12 part of the complex and significant bioactivity of the pharmacological part of the complex.

Methods for administering oligonucleotide or polynucleotide agents of the invention are also well known in the art (see Dass, 2002, J Pharm Pharmacol. 54 (1): 3-27; Dass, 2001, Drug Deliv. 8 (4): 191- 213; Lebedeva et al., 2000, Eur J Pharm Blopharm. 50 (1): 101-19; Pierce et al., 2005, Mini Rev Med Chem. 5 (1): 41-55; Lysik and WuPong, 2003, J Pharm Sci. 2003 2 (8): 1-559-73; Dass, 2004, Biotechnol Appl Biochem. 40 (Pt 2): 113-22; Medina, 2004, Curr Pharm Des. 10 (24): 2981-9 ).

The composition of the second aspect of the invention may further comprise at least one other agent.

Said additional agent may be an anti-inflammatory agent that includes but is not limited to a non-steroidal anti-inflammatory agent (NSAID), a disease-modifying anti-rheumatic drug (DMARD), a statin (including HMG-CoA reductase inhibitors such as simvastatin), a biological agent (biological products), a steroid, an immunosuppressive agent, a salicylate and / or a microbicidal agent. Non-steroidal anti-inflammatory agents include antimetabolite agents (such as methotrexate) and anti-inflammatory gold agents (including gold sodium thiomalate, aurothiomalate or gold salts, such as auranofin). Biological products include anti TNF agents (including adalimumab, etanercept, infliximab, anti-IL-1 reagents, anti-IL-6 reagents, B-lymphocyte reagents (retoximab), T-lymphocyte reagents (anti-CD4 antibodies), anti-IL-15 reagents , anti CLTA4 reagents, anti RAGE reagents), antibodies, soluble receptors, receptor binding proteins, cytokine binding proteins, mutant proteins with altered or attenuated functions, RNAi, polynucleotide aptamers, antisense oligonucleotides or omega 3 fatty acids. Steroids (also known as corticosteroids) include cortisone, prednisolone or dexamethasone. Immunosuppressive agents include cyclosporine, FK506, rapamycin, mycophenolic acid. Salicylates include aspirin, sodium salicylate, choline salicylate and magnesium salicylate. Microbicidal agents include quinine and chloroquine. For example, the agent can be administered in combination with one or more of an NSAID, DMARD or immunosuppressant.

An agent or composition as defined herein for use as a medicament is also described herein.

In a third aspect of the invention an agent or composition is provided as defined in the first or second aspects of the invention for use in the treatment of a chronic inflammatory condition, in which the chronic inflammatory response is associated with rheumatoid arthritis. (RA), inflammatory bowel diseases, atherosclerosis and / or psoriasis.

In a fourth aspect of the invention there is provided the use of an agent or composition as defined in the first

or second aspects of the invention in the preparation of a medicament for the treatment of a chronic inflammatory condition, in which the chronic inflammatory response is associated with rheumatoid arthritis (RA), inflammatory bowel diseases, atherosclerosis and / or psoriasis.

Also described herein is a method of treating a chronic inflammatory condition that comprises administering to an individual an effective amount of an agent or composition as defined herein.

The agent, composition, use or method as defined herein may be related to the treatment of a chronic inflammatory condition in which the condition is associated with any condition associated with inappropriate inflammation. Such conditions include, but are not limited to, rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel diseases, wounds that are not cured, multiple sclerosis, cancer, atherosclerosis, sjogren's disease, diabetes, lupus erythematosus (including systemic lupus erythematosus), asthma, fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UV damage and psoriasis.

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Also described herein is a kit of parts for performing the methods described herein comprising:

<dl><dt>(i)</dt><dd> one or more cells </dd></dl>

<dl><dt>(ii)</dt><dd> a control sample of one or more cells </dd></dl>

(iii) a sample of tenascin C

(iv) instructions for use

The kit may optionally comprise:

(v) a candidate agent.

The kit may also optionally comprise

(vi) means to determine the effect of a candidate agent on the activity of tenascin C or chronic inflammation.

In a fifth aspect of the invention there is provided a kit of parts comprising:

<dl><dt>(i)</dt><dd> an agent or composition as defined in the first or second aspects of the invention </dd></dl>

<dl><dt>(ii)</dt><dd> means of administration </dd></dl>

(iii) instructions for use

The kit of the fifth aspect of the invention may optionally further comprise

(iv) at least one other agent.

Definitions

By "inflammation" is included the meaning of local accumulation of fluid, plasma proteins and white blood cells that is initiated by tissue injury, infection or a local immune response.

By "acute inflammation" is included the meaning of the initial stages (onset) of inflammation and the short-term transient inflammatory response immediately after the injury, infection or local immune response. Normally, acute inflammation resolves rapidly, lasting from several minutes to no more than several days.

By "chronic inflammation" the meaning of persistent and / or unresolved inflammation is included. It is frequently associated with inappropriate destruction of healthy tissue. This can be progressive and last for a period of weeks.

or more. Chronic inflammation is usually associated with infection or persistent disease including, but not limited to, autoimmune conditions.

By "chronic inflammation of the joint" the meaning of persistent inflammation that is progressive and does not remit for a period of weeks to months is included, resulting in distortion of the affected joint and radiographic evidence of destruction of cartilage and bone as seen in human disease (Kelly, Harris, Ruddy and Sledge, Textbook of Rheumatology 4th Edition).

In experimental murine models, chronic inflammation of the joint is characterized by inflammation that does not diminish and causes inappropriate tissue destruction, even for a relatively short period of time. This is characterized (and can be identified) histologically by the prolonged presence of inflammatory cells in the synovium and joint space, chondrocyte death and erosion of the cartilage and bone.

An "agent" means all chemical entities, for example oligonucleotides, polynucleotides, polypeptides, peptidomimetics and small compounds.

By "fragment" is meant at least 10 nucleotides, for example at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.

By "variant" is meant that the nucleotide sequence shares at least 90% sequence identity with the sequence of interest of full length, for example at least 50%, 55%, 60%, 65%, 70%, 75% , 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity.

The percentage of sequence identity between two polynucleotides can be determined using suitable computer programs, for example the GAP program of the Genetic Computing Group of the University of Wisconsin and it will be appreciated that the percentage of identity is calculated in relation to polynucleotides whose sequences have been aligned optimally.

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Alignment can alternatively be carried out using the Clustal W program (as described in Thompson et al., 1994, Nuc. Acid Res. 22: 4673-4680).

The parameters used can be the following:

Quick peer alignment parameters: K-tuple size (word); 1, window size; 5, gap penalty; 3, number of upper diagonals; 5. Scoring method: x percent.

Multiple alignment parameters: open hole penalty; 10, gap extension penalty; 0.05

Score matrix: BLOSUM.

Alternatively, the BESTFIT program can be used to determine local sequence alignments.

By "antibody" are meant substantially intact antibody molecules, as well as chimeric antibodies, humanized antibodies, human antibodies (in which at least one amino acid is mutated in relation to naturally occurring human antibodies), single chain antibodies, bispecific antibodies, chains heavy antibodies, light chains of antibodies, homodimers and heterodimers of heavy and / or light chains of antibodies, and antigen binding fragments and derivatives thereof.

By "antigen binding fragment" is meant a functional fragment of an antibody that is capable of binding with tenascin C.

The term "subject" means all animals including humans. Examples of subjects include humans, cows, dogs, cats, goats, sheep and pigs. The term "patient" means a subject who has a disorder that needs treatment.

As used herein, "pharmaceutical formulation" means a therapeutically effective formulation according to the invention.

An "effective therapeutic amount" or "effective amount" or "therapeutic amount", as used herein, refers to the amount that provides a therapeutic effect for a given condition and administration regimen. This is a predetermined amount of active material that has been calculated to produce a desired therapeutic effect in association with the required additive and diluent, ie a transporter or delivery vehicle. In addition, it is intended to mean an amount sufficient to reduce and more preferably prevent a clinically significant deficit in host activity, function and response. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in a host. As appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity. Suitable dosage amounts may contain a predetermined amount of active composition calculated to produce the desired therapeutic effect in association with the required diluent. In the methods and use for manufacturing compositions of the invention, a therapeutically effective amount of the active component is provided. A therapeutically effective amount can be determined by the usual expert medical or veterinary worker based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art.

Examples representing an aspect of the invention will now be described with reference to the following figures in which:

Figure 1. Accelerated resolution of acute inflammation in mice deficient in tenascin C.

(a) Swelling of the paw in wild-type (+ / +) mice (white bars) and without tenascin C (- / -) (black bars) over time after zimosan injection. The data are shown as the average increase in the diameter of the leg compared to the diameter of the leg before injection +/- ETM (n = 24 mice per genotype). ** = p &lt;0.01. (be) Representative sections of the ankle joint of wild-type mice (b, c) and without tenascin C (d, e) 4 days after injection of zymosan, stained with hematoxylin and eosin (b, d) and safranine -O (c, e). The boxes highlight the synovium of the joint (s) and cartilage proteoglycan (cp). Increase x10. Quantification of joint inflammation (f) and chondrocyte death (g) in knee joints 4 days after injection with zymosan of wild-type mice (white bars) and mice without tenascin C (black bars). Data are expressed as the mean (+/- DT) (n = 24 mice per genotype). * = p &lt;0.05.

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Figure 2. Synovial inflammation is induced in tenascin C deficient mice after antigen injection.

(ab, g) Representative sections of the knee joint of wild-type mice injected with simulation. (cf, hi) Representative sections of the knee joint of wild-type (c, d, h) or non-tenascin C (e, f, i) mice 24 hours after intra-articular mBSA injection. The infiltration of inflammatory cells in the capsule, meniscus and joint space of both wild-type and non-tenascin C mice is highlighted by (cap), (M) and (J) respectively. (S) Highlights the healthy synovium of mice injected with simulation that is not more than 1-3 cells thick along the entire bone surface and (ST) highlights the synoviums of wild-type and non-tenascin C mice that are both significantly thickened. The sections are stained with hematoxylin and eosin (a, c, e, g, h, i) and safranine O (b, d, f). Increase x10 (af) or x40 (gi). (n = 5 mice per genotype).

Figure 3. Synovial inflammation decreases rapidly in mice deficient in tenascin C.

Representative sections of the knee joint of wild-type mice (a, b, f) or without tenascin C (c, d, e) 3 days after intraarticular injection of mBSA. (a, c) The line highlights increased capsule inflammation in wild-type mice compared to mice without tenascin C. (b, d) (cp) highlights increased cartilage proteoglycan loss in wild-type mice compared to mice without tenascin C. (e, f) Significant synovial hyperplasia (line), cell and fibrin deposits in the joint space (arrow) and invasion of pannus (arrow heads) are observed in wild-type mice compared to mice without tenascin C. The sections are stained with hematoxylin and eosin (a, c, e, f) and safranine O (b, d). Increase x10 (ad) or x20 (ef). (n = 5 mice per genotype).

Figure 4. Tenascin C-deficient mice are protected from tissue destruction during antigen-induced arthritis.

(ab) Representative sections of the knee joint of wild-type mice 7 days after intraarticular injection of mBSA, stained with hematoxylin and eosin (a) and safranine-O (b). Increase x10. (n = 24 mice per genotype). The arrowhead highlights the area of ​​bone erosion. The arrow highlights the invasion of pannus in the articular cartilage. (cd) Representative sections of the knee joint of mice without type tenascin-C 7 days after intraarticular injection of mBSA, stained with hematoxylin and eosin (c) and safranine-O (d). Increase x10. (n = 24 mice per genotype). J highlights the joint space and AC the intact articular cartilage. (e) Histological score of inflammation of the knee joint 24 hours, 3 days and 7 days after mBSA injection of wild-type mice (white bars) and mice without tenascin C (black bars). The data represent the mean +/- DT (n = 5 per genotype (24h, 3d) or 24 per genotype (7d)). (f) Quantification of chondrocyte death, cartilage surface erosion and bone erosion after mBSA injection into knee joints of wild-type mice (white bars) and mice without tenascin C (black bars). Chondrocyte death is shown at 24 hours, 3 days and 7 days, and cartilage surface erosion and bone erosion at 7 days. The data represent the mean +/- DT (n = 5 per genotype (24h, 3d) or 24 per genotype (7d)).

Figure 5. Tenascin C induces synthesis of TNF-, IL-6 and IL-8 in primary human macrophages and synovial fibroblasts of RA.

(ab) Primary human macrophages (a) and synovial fibroblasts with RA (b) were not stimulated (without addition) or stimulated with LPS (1 ng / ml (a) or 10 ng / ml (b)) or tenascin C recombinant (1.0 --M - 1.0 nM) for 24 hours. The data shown are the average of triplicate values ​​(+/- DT) of one of three representative experiments. (c) Primary human macrophages were not stimulated (without addition) or stimulated with LPS (1 ng / ml) or recombinant tenascin C (1.0 µM) for 24 hours. (-) indicates that the cells were pre-incubated only with medium. (P) Cells were pre-incubated with 25 µg / ml polymyxin B for 30 minutes before stimulation. (H) The cells were incubated with medium without addition or containing LPS or tenascin C which was boiled for 15 minutes before addition to the cells. The data shown are the measurement of triplicate values ​​(+/- DT) of one of three representative experiments.

Figure 6. The FBG domain of tenascin C mediates the stimulation of cytokine synthesis in vivo and in vitro.

(a) Primary human macrophages were not stimulated (without addition) or stimulated with LPS (1 ng / ml), recombinant tenascin C (TNC) or tenascin C 1.0 M domains (TA, EGF-L, TNIII1 -5, TNIII1-3, TNIII3-5, TNIII5-7, TNIII6-8 and FBG) for 24 hours. The data shown are the average of triplicate values ​​(+/- DT) of one of three representative experiments. (b) Synovial membrane cells with RA were not stimulated (without addition) or stimulated with LPS (10 ng / ml) or recombinant FBG (1.0 - 0.01 µM) for 24 hours. The data shown are the average percentage change in cytokine levels compared to unstimulated cells (+/- ETM) of five different patients. (ch) Sections

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Representative of the knee joint of wild-type mice 3 days after intra-articular injection of PBS (ce) or 1 g of FBG (fh). The sections are stained with hematoxylin and eosin (c, d, f, g) or Safranine-O (e, h). Increase x10 (c, f) or x25 (d, e, g, h) (n = 5 mice per genotype). (i) Quantification of joint inflammation, bone erosion, cartilage surface erosion and chondrocyte death in the knee joints of wild-type mice 3 days after intra-articular injection of PBS (black bars) or 1  g of FBG (white bars). The data represent the mean +/- DT (n = 5 per genotype).

Figure 7. FBG-mediated cytokine synthesis is dependent on MyD88.

(a) Human RA synovial fibroblasts were not infected, infected with adenovirus expressing GFP only (AdGFP) or infected with adenovirus expressing dominant negative MyD88 (AdMyD88dn). The cells were not stimulated, stimulated with LPS (10 ng / ml) or stimulated with FBG (1 µM) for 24 hours. The data shown are the average of three independent experiments (+/- ETM). (b) Mouse embryonic fibroblasts isolated from wild-type (+ / +) or MyD88-deficient (- / -) mice were not stimulated (-) or stimulated with PAM3 (100 ng / ml), LPS (100 ng / ml), TNF (100 ng / ml), IL-1 (5 ng / ml) and FBG (1 M) for 24 hours. The data shown are the average of three independent experiments (+/- ETM).

Figure 8. FBG-mediated cytokine synthesis is dependent on TLR4 but does not require CD14 or MD

2.

(a) Primary human macrophages were pre-incubated with medium only or medium containing function blocking antibodies for TLR2 (10 µg / ml), TLR4 (25 µg / ml) or isotype control antibodies (25 µg / ml) for 30 minutes before stimulation. The cells were not stimulated, or stimulated with LPS (1 ng / ml), FBG (1 M) or PAM3 (10 ng / ml) for 24 hours. The data shown are the average of three independent experiments (+/- ETM). (b) Mouse embryonic fibroblasts isolated from wild-type mice, deficient in TLR2 (TLR2 - / -) or TLR4 (TLR4 - / -) were not stimulated or stimulated with PAM3 (100 ng / ml), LPS (100 ng / ml), IL-1 (5 ng / ml) and FBG (1 M) for 24 hours. The data shown are the average of three independent experiments (+/- ETM). (c) Bone marrow derived macrophages isolated from wild-type mice, deficient in TLR2 (TLR2 - / -) or TLR4 (TLR4 - / -) were not stimulated or stimulated with PAM3 (100 ng / ml), LPS ( 100 ng / ml) or FBG (1 M) for 24 hours. The data shown are the average of three independent experiments (+/- ETM). (d) Human macrophages were pre-incubated without inhibitor, with msbB LPS 1 g / ml or anti-CD14 antibody 10 /g / ml for 30 minutes before stimulation with LPS (1 ng / ml), FBG (1 M) or PAM3 (10 ng / ml) for 24 hours. The data shown are the average of three independent experiments (+/- ETM).

Figure 9. Swelling of the leg over time after injection of zymosan.

Representative images of the legs of mice without tenascin C not injected (a, e) (diameter 1.6 mm), mice without tenascin C 24 hours (d, f) (diameter 2.5 mm) and 4d (b, h) (diameter 1.7 mm) after injection of zymosan and wild-type mice 4 days after injection of zymosan (c, g) (diameter 2.1 mm).

Figure 10. Synthesis of recombinant proteins.

(a) Tenascin C monomer domain structure comprising different domains, including assembly domain (TA), 14 and a half repetitions of type EGF (EGF-L), 17 repetitions of type III fibronectin (TNIII) (8 expressed constitutively (1-8) and 9 that can be cut and spliced ​​alternately, and a fibrinogen-like globule (FBG). (b) The regions covered by the recombinant proteins that were synthesized, the corresponding amino acid residues and the molecular weight of each protein.

Figure 11. Protein purity analysis.

Silver-stained gel showing 1 µg of each recombinant protein analyzed by SDS-PAGE under reducing conditions. Lane: 1 (TA), 2 (EGF-L), 3 (TNIII1-5), 4 (TNIII5-7), 5 (TNIII6-8), 6 (TNIII1-3), 7 (TNIII3-6) and 8 (FBG)

Figure 12. FBG-mediated joint inflammation in vivo requires expression of TLR4.

Representative sections of the knee joint of mice without TLR2 (a) and TLR4 (b) 3 days after intra-articular injection of 1 g of FBG. Sections are stained with hematoxylin and eosin. Increase x10 (n = 5 mice per genotype). (c) Quantification of joint inflammation, bone erosion, erosion of the cartilage surface and death of chondrocytes in the knee joints of mice without TLR2 (white bars) and TLR4 (black bars) 3 days after 1 g intra-articular injection of FBG. The data represent the mean +/- DT (n = 5 per genotype).

Figure 13. Amino acid sequence of human tenascin C and its domains

Figure 14. Nucleotide sequence of human tenascin C

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Figure 15. Synthesis of TNF in response to specific FBG peptides.

Synthesis of TNF by membrane cultures with RA incubated for 24 hours without addition or with 100 µM of each FBG peptide (P1, P3-P9).

5 Figure 16. Synthesis of TNF and IL8 in response to various concentrations of specific FBG peptides.

Synthesis of TNF and IL8 by membrane cultures with RA incubated for 24 hours without additions or with 25, 100

or 250 µM of FBG peptide.

10 Figure 17. Synthesis of IL8 in response to LPS, complete FBG domain or specific FBG peptides. Synthesis of IL8 by macrophages after 24 hours of incubation without addition, with 1 ng / ml LPS, 1 M complete FBG domain (FBG) or 1 or 20 FM FBG peptides (P1, P3-P9).

Figure 18. Synthesis of IL8 and TNF in response to LPS and FBG after preincubation with peptides of

fifteen FBG Synthesis of TNF and IL8 by macrophages after 24 hours of incubation without addition, with 1 ng / ml LPS or 1 M complete FBG domain (FBG) with or without preincubation with 20 M FBG peptides.

Figure 19. Synthesis of IL8 and TNF in response to siRNA directed to tenascin C.

twenty Tenascin C mRNA levels in fibroblasts with RA transfected with luciferase-specific siRNA (control) or with tenacin C-directed siRNA: oligo 1 (ip 1), oligo 2 (ip 2) or a combination of oligos 1 + 2 (ip 1 + 2). Synthesis of IL6 in fibroblasts with RA transfected with luciferase siRNA (control) or with a combination of oligos directed to tenascin C 1 + 2 (siRNA) in the presence or absence of 10 ng / ml LPS for 24 hours.

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Example 1 - General Methods

Reagents

30 The zymosan, Freund's methylated BSA and complete adjuvant, anti FLAG M2 antibody (mouse monoclonal antibody), blasticidine and isotype control antibodies (IgG2a, mouse IgG1) were from Sigma-Aldrich (Dorset, UK). Hypnorm was from VetaPharma Ltd. (Leeds, United Kingdom). Limulus amaebocyte's lysate test was from the Associates of Cape Cod (Liverpool, United Kingdom). The wild-type human embryonic kidney cells (HEK293-EBNA) were from Invitrogen (Groningen, The Netherlands). M-CSF and IL-1 were from PeproTech (Neuilly-Sur

35 Seine, France). DMEM, RPMI 1640, fetal bovine serum (FBS), penicillin / streptomycin, PSA and ant-mercaptoethanol antifungal solution were from PAA Laboratories (Yeovil, United Kingdom). HEK293 cell lines stably expressing human TLR2 and TLR4 / CD14 / MD-2, polymyxin B, msbB LPS and TLR2 function blocking antibodies (Clone: ​​TL2.1 Isotype: Mouse IgG2a) and TLR4 (Clone: ​​HTA125 Isotype: mouse IgG2a) were from Invivogen (Calne, United Kingdom). Purified Escherichia coli LPS in phenol-chloroform (rough and smooth) and

40 Pam3Cys-Ser-Lys4 (Pam3C) were from Alexis (Birmingham, United Kingdom). TNF- and murine IL1 receptor antagonist (IL-1ra-IL-1 F3) were from R&amp;D Systems (Abingdon, UK). The anti-CD14 function blocking antibodies (Isotype: mouse IgG1) were from Abcam (Cambridge, United Kingdom). The human and murine TNF-, IL-6 and IL-8 ELISAs were from Pharmingen (Oxford, United Kingdom).

Four. Five Tenascin C full length purification

To ensure that the production of cytokines was not attributed to bacterial contaminants such as LPS and LPS-associated molecules, recombinant full-length human tenascin C was purified from conditioned medium of the human HEK293 mammalian cell line transfected with his labeled human tenascin C at

fifty pCEP-pu vector as described (Lange (2007)). Tenascin C was purified to homogeneity as described (Lange (2007) and determined to be free of LPS contamination using the Limulus amaebocyte lysate test according to the manufacturer's instructions.

Synthesis of recombinant proteins

55 Proteins corresponding to each tenascin C domain (TA, EGF-L, various repeats of TNIII and FBG) were synthesized and purified. See Example 2.

Measurement of contamination with LPS in recombinant proteins

60 To determine the levels of LPS in each recombinant protein, the Limulus amaebocyte lysate assay was used according to the manufacturer's instructions (sensitivity 0.7  0.5 pg of LPS per mg protein). All recombinant proteins used in this study had levels of LPS that were less than 10 pg / ml.

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Adenoviral vectors and their spread

Recombinant, replication-deficient adenoviral vectors encoding wild-type MyD88 (AdMyD88wt), dominant negative forms of MyD88 (AdMyD88dn) and GFP control (AdGFP) were internally clogged. There is a description of the synthesis of these viruses in Andreakos (2004). All viruses used in this study have E1 / E3 suppression, belong to the Ad5 serotype. Viruses were propagated in 293 human embryonic kidney cells, purified by ultracentrifugation through two gradients of cesium chloride, and their viral titers were determined by plaque assay as previously described (Sacre (2007)).

Animals

Professor Charles French-Constant (University of Edinburgh, United Kingdom) provided homozygous tenascin C deficient mice of the original strain described by Saga (1992) in 129 / sv an inbred strain of mice with a white belly bottom and agouti appearance . 129 / sv wild-type, inbred, congenital mice of the same age were obtained from Charles River (Margate, United Kingdom). All 129 / sv wild-type and tenascin C-deficient mice were male and between 8 and 10 weeks of age at the time of experimentation.

Homozygous TLR2 and TLR4 deficient mice were obtained in a C57BL / 6 background (an inbred strain of mice with a black coat) from B&amp;K Universal (Hull, United Kingdom) Hoshino (1999) and Takeuchi (1999). Homozygous MyD88-deficient mice were provided in a C57BL / 6 fund by the Sanger Institute (Cambridge, United Kingdom). C57B / L6 wild-type congenital inbred mice of the same age were obtained from Charles River (Margate, United Kingdom). For the isolation of mouse embryo fibroblasts, an 8-10 week old female was mated with two 8-10 week old males. For the isolation of macrophages derived from bone marrow the mice were female and between 10 and 12 weeks of age at the time of experimentation.

All animals were fed with conventional rodent feed and water at will, and were housed (&lt;6 mice / cage) in sawdust-cages in an air-conditioned environment with 12-hour light / dark cycles. All animal procedures were approved by the institutional ethics committee.

Statistical Methods

Mean, DT, ETM and statistical tests were calculated using GraphPad version 3 (GraphPad Software Inc., San Diego, CA). The means of multiple groups were analyzed by one-way analysis of variance, followed by the Dunnett's Multiple Comparisons test, when appropriate. T-test was used for unrelated samples for experiments involving only two groups.

Example 2 - Synthesis of recombinant proteins

Proteins corresponding to each tenascin C domain (TA, EGF-L, various repeats of TNIII and FBG) were synthesized and purified. Recombinant synthesized proteins are depicted in Figure 9.

Reagents

The Pfu Turbo polymerase was from Stratagene (Amsterdam, The Netherlands). Easy mix 50 PCR tubes were from Molecular Bioproducts (Lutterworth, United Kingdom). The RNeasy kits and Ni2 + -NTA-agarose columns were from Qiagen (Crawley, United Kingdom). Blunt pCR vector, pCEP4 plasmid vector, human embryonic kidney cells (HEK293-EBNA) and 4-12% Bis-Tris gradient gels were from Invitrogen (Groningen, The Netherlands). The vector pET32b and BL21 (DE3) Rosetta cells were from Novagen (Kent, United Kingdom). The HiTrap Q columns, HiTrap S columns, Sephacryl S500 HR column and heparin sepharose columns were from Amersham (Buckinghamshire, United Kingdom).

Restriction enzymes were obtained from New England BioLabs (Hitchin, United Kingdom). DMEM, fetal bovine serum (FBS) and penicillin / streptomycin were from PAA laboratories (Yeovil, United Kingdom). FuGENE6 transfection reagent was from Roche Applied Science (Basel, Switzerland).

The anti FLAG M2 antibody (mouse monoclonal antibody), anti FLAG M2-agarose, FLAG peptide were from Sigma-Aldrich (Dorset, United Kingdom). The anti-tetra-his antibody (mouse monoclonal antibody) was from Qiagen (Crawley, United Kingdom). Goat anti IgG (mouse IgG) conjugated with alkaline phosphatase and Western Blue stabilized substrate for alkaline phosphatase were from Promega (Southampton, UK). The Precision Protein Patterns for SDS-PAGE were from BioRad (Hemel Hempstead, United Kingdom).

Primer Design

Domain boundaries were determined using alignments published in the human tenascin C sequence (Siri (1991) reference number P24821 (Swiss-Prot)). To clone each domain the inventors designed

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PCR primers in which both direct and reverse primers contained 18-21 bases corresponding to the 5 'and 3' terminal sequences of the necessary coding sequence. The direct primer contained a restriction site Nde1, followed by a his N terminal marker, immediately before the coding sequence. The final 3 bases of the Nde1 site form the ATC methionine start codon. The reverse primer included a TTA stop codon immediately after the coding sequence, followed by a BamH1 site or a Kpn1 to allow unidirectional cloning to pET32b expression vectors.

Table 1

<dl><dt>Protein name </dt><dd>Direct primer Reverse primer </dd></dl>

<dl><dt>TA </dt><dd /></dl>

<dl><dt>EGF-L </dt><dd /></dl>

<dl><dt>TN1-5 </dt><dd /></dl>

<dl><dt>TN1-3 </dt><dd /></dl>

<dl><dt>TN3-5 </dt><dd /></dl>

<dl><dt>TN5-7 </dt><dd /></dl>

<dl><dt>TN6-8 </dt><dd /></dl>

<dl><dt>FBG </dt><dd /></dl>

10 All previous primers are written from 5 'to 3'. Flag sequences are in bold, His markers (CATCATCATCATCATCAT) are underlined, and restriction enzyme cleavage sites (CATATG = site Nde1, GGATCC = BamH1, GGTACC = site Kpn1) are italic bold.

PCR

fifteen PCR amplification was carried out using 10 pmol / µl of each primer, 1 µg of template, 5 µl of DMSO and 1.25 units of Pfu Turbo polymerase in a final volume of 25 µl. This was added to the buffer and dNTP in Easy tubes.

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mix 50. The template used for all reactions was cDNA prepared from U87MG human glioma cells using RNA isolated with RNeasy kits. R eacción cyclized temperatures 40 times with denaturation, annealing and elongation 95 ° C, 55-65 ° C (depending on the melting temperature (Tm) of the primers) and 72 ° C respectively.

Cloning

PCR products were ligated into Blunt pCR vectors and sequenced to ensure that no PCR errors had been introduced. Clones that had no errors or silent mutations were selected. Inserts were then ligated into pET32b using restriction sites Nde1 and BamH1 genetically engineered into primers (TN5-7 and TN6-8). Human tenascin C has internal BamH1 sites within the TA domain (position 494) and TNIII2 (position 2509). TA and TN1-8 were therefore cloned using the Nde1 site in the DIR primer and the Kpn1 site in the cloning site of pCRBlunt. Human tenascin C does not contain internal Kpn1 sites. TN1-5, TN1-3 and TN3-5 were cloned using Nde1 and Kpn1 sites in the primers. FBG contains an internal Nde1 site (position 6439) and was therefore cloned using a two-stage digestion linkage with Nde1 and BamH1, followed by Nde1 digestion. (The positions refer to sites within the tenascin C full length nucleotide sequence, provided in Figure 14).

Bacterial growth, induction and lysis

Plasmids were transformed into BL21 (DE3) Rosetta cells, grown in 3 l of Luria-Bertani medium containing 50 queg / ml carbenicillin and induced with 1 mM isopropyl--D-thiogalactopyranoside. After 3 hours, the cells were collected by centrifugation at 4,000 rpm for 20 minutes, washed twice with ice wash buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl and 1 mM EDTA) and lysed With a french press. Inclusion bodies were collected by centrifugation at 12,000 rpm for 20 minutes at 4 ° C. With the exception of TA and FBG proteins were completely located in the supernatant. Recombinant TA and FBG proteins were included from inclusion bodies with 6 M guanidine hydrochloride, 50 mM Tris-HCl, pH 8.0 and 10 mM camercaptoethanol at room temperature with constant stirring for 2 hours.

Bacterial protein purification

The solution containing recombinant protein was applied to a Ni2 + -NTA-agarose column and washed with 50 mM Tris-HCl, pH 8.0 containing 20 mM imidazole. The column was subsequently washed with 50 mM Tris-HCl, pH 8.0 and the protein was eluted with 50 mM Tris-HCl, pH 8.0 containing 60 mM imidazole. For TA and FBG each wash and elution buffer contained 6 M guanidine hydrochloride. After chromatography with Ni TA and FBG they did not require further purification. TN1-3 and TN6-8 were further purified by anion exchange chromatography using a HiTrap Q column, TN1-5, TN3-5 and TN5-7 by cation exchange chromatography using a HiTrap S column and TN1-8 using a HiTrap column S followed by gel filtration using a Sephacryl S500 HR column.

Insoluble protein refolding

TA and FBG were refolded by diluting up to 20 µg / ml with 50 mM Tris-HCl, pH 8.0 containing 6 M guanidine hydrochloride and then treating with 20 mM cystamine with stirring for 16 hours at 4 ° C. The solution was then dialyzed twice against 15 volumes of 50 mM Tris-HCl, pH 8.0 containing 150 mM NaCl, 10 mM CaCl2, 5 mM camercaptoethanol and 1 mM 2-hydroxyethyl disulfide for 24 hours at 4 ° C, twice against 20 mM Tris-HCl, pH 8.0 for 8 hours at 4 ° C and then centrifuged at 12,000 rpm for 30 minutes at 4 ° C. Refolding was evaluated by size shifts using SDS PAGE under reducing and non-reducing conditions. Protein activity was confirmed by polymerization of the TA domain and binding of FBG with heparin sepharose columns.

Synthesis of the EGF-L domain using mammalian cells

Initial attempts to express and purify the EGF-L repeat region using an E. coli expression system were unsuccessful. This is most likely attributable to the difficulty in achieving protein folding due to a total of 91 cysteines in this region. Therefore, TNF-type EGF domains were expressed using HEK293 cells.

Two PCR reactions were carried out. The first PCR product consisted of a KpnI restriction enzyme site, a Kozak sequence followed by the TN-C signal sequence. The second PCR product consisted of a FLAG peptide, the EGF type domain sequence, followed by a histidine marker and a BamH1 restriction enzyme sequence.

The two PCR products were linked together as described in Ho (1989). PCR reactions were carried out as described above. The complete construct was cloned into the PCR Blunt vector and sequenced. It was then subcloned into the vector pCEP4. The DNA was transferred to HEK293 cells using Fugene and was

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selected cells for hygromycin resistance (200 µg / ml) in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (v / v), penicillin (100 units / ml) and streptomycin (100 units / ml) 2 liters of conditioned medium (collected after cells have been cultured in medium) were collected and pooled from stably transfected cells. The pooled conditioned medium (2 liters) was centrifuged at 3000 rpm to separate cell debris from the medium.

The medium was then applied to an anti FLAG column. Material was collected in 50 ml fractions for continuous flow. The column was washed with 10 column volumes of 1M NaCl, 50 mM Tris-HCl, pH 7.5 and then washed with 10 column volumes of 60% isopropanol to ensure removal of LPS. The column was then washed with 50 mM Tris-HCl buffer, pH 7.5 and finally the protein was eluted using 200 µg / ml FLAG peptide in 50 mM Tris-HCl buffer, pH 7.5.

Protein purity analysis

Each protein was dialyzed against 1000 volumes of 150 mM NaCl and 50 mM Tris pH 7.5. Protein purity was analyzed by SDS-PAGE under reducing conditions. To do this, 1 µg of each purified recombinant protein was processed on a 4-12% Bis-Tris gradient gel and the gel was then stained with silver to demonstrate a single band (Figure 10). Western blot analysis was also carried out. The proteins separated by SDS-PAGE were electrotransferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% BSA in Tris buffered saline and then incubated with primary antibodies that recognized FLAG M2 antibodies (1: 2000 dilution) (EGF-L) or tetra-his (1: 2000) (all the other proteins). The transfer was then incubated with secondary antibody conjugated with alkaline phosphatase and the protein bands were visualized using Western Blue stabilized substrate so the genes show a single specific band recognized by each antibody at the expected Pm (not shown).

Example 3 - Animal models

Zymosan-induced arthritis

Zymosan-induced arthritis (ZIA) was induced in mice deficient in tenascin C and wild-type by injection of zymosan (Saccharomyces cerevisiae), as described in Keystone (1977). Zymosan was prepared by dissolving 15 mg of zymosan in 1 ml of sterile PBS. The solution was boiled twice and sonicated. Mice were anesthetized by intraperitoneal injection of 150 µl of Hypnorm diluted 1:10 in sterile water, then zymosan (10 µl) was injected into the right plantar pad (d = 0).

Control mice received a 10 µl injection of PBS alone or were not injected. For macroscopic evaluation of arthritis, the thickness of each hind leg was measured daily with microcalibrators (Kroeplin, Schluchlem, Germany) and the diameter was expressed as a mean for each hind leg swollen by mouse.

After completing the experiment (day = 4), the mice were sacrificed and the hind legs fixed in 10% buffered formalin (v / v), decalcified with 10% EDTA and processed with paraffin.

Antigen-induced arthritis

Antigen-induced arthritis (AIA) was induced in tenascin C-deficient and wild-type mice as previously described by Brackertz (1977). Briefly, on day 0 the mice were anesthetized by intraperitoneal injection of 150 µl of Hypnorm diluted 1:10 in sterile water, then immunized with 200 µg of methylated BSA. MBSA was emulsified in 0.2 ml of Freund's complete adjuvant and injected intradermally at the base of the tail.

On day 7, arthritis was induced by intraarticular injection of mBSA (100 µg in 10 µl of sterile PBS) into the right knee joint using a sterile 33 gauge microcanula. Control mice received an injection of 10 µl of PBS alone or were not injected.

On day 14, mice were sacrificed, knee joints were excised and fixed in 10% buffered formalin (volume / volume) were decalcified, with 10% EDTA and processed with paraffin.

FBG injection

Wild-type mice were anesthetized by intraperitoneal injection of 150 µl of Hypnorm diluted 1:10 in sterile water and then injected 100 ng, 1 or 3 µg of FBG into 10 µl of sterile PBS in the joint of the right knee using a sterile 33 gauge microcannula. Control mice received a 10 µl injection of PBS alone or were not injected.

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On days 3 and 7, mice were sacrificed, knee joints were excised and fixed in 10% buffered formalin (volume / volume), decalcified, with 10% EDTA and processed with paraffin.

Histology of the knee joints

Coronal tissue sections (4 4m) were cut at 7 depths throughout the joint; at 80 m away and stained with hematoxylin and eosin or Safranin-O to assess the pathology of the joint. Histopathological changes were scored using the following parameters as described in Van Lent (2006).

Inflammation (the inflow of inflammatory cells into the synovium (infiltrate) and the joint cavity (exudates)), was classified using an arbitrary scale from 0 (without inflammation) to 3 (severe inflammation). Chondrocyte death was determined as the percentage of cartilage area that contained empty lagoons in relation to the total area. Erosion or cartilage surface was determined as the amount of cartilage lost in relation to the total cartilage area. Bone destruction in 10 different areas of the total knee joint section was determined. The destruction was classified on a scale of 0 (no damage) to 3 (complete loss of bone structure). A histological analysis was performed by a blind investigator to the experimental groups. The average score for each animal in an experimental group was calculated by averaging histopathological scores at at least 5 depths of section per joint.

Results

Zymosan-induced joint inflammation is not maintained in mice deficient in tenascin C

Zimosan injection into the plantar pad was used to induce acute synovitis in mice. The wild-type mice showed rapid swelling of the leg reaching a maximum leg diameter at 24 hours (2.56 mm, an increase of 62% of the starting diameter of the leg). This was maintained for 24 more hours. After 2 days the diameter of the leg was reduced but the legs remained swollen until 4 days (2.08 mm, an increase of 32%) (Figure 1a). Tenascin C-deficient mice showed a similar degree of swelling of the legs to wild-type mice 24 hours after injection (2.41 mm, a 57% increase in the starting diameter of the leg). However, swelling in mice without tenascin C decreased more rapidly than in wild-type mice; the diameter of the leg was significantly reduced at 2 days and had been reduced to 1.7 mm (an increase of only 11%) at 4 days (Figure 1a). On day 4 after injection, the legs of the wild-type mice were still visibly swollen and red, while the legs of mice without tenascin C were not visibly swollen or red and resembled non-injected legs (Figure 9).

This difference was reflected histologically at 4 days. Synoviums of wild-type mice were significantly inflamed and showed cell infiltration and loss of cartilage proteoglycans was observed (Figure 1b, c). In contrast, the synovium of tenascin C-deficient mice showed no synovitis, cell infiltration or loss of cartilage proteoglycans (Figure 1d, e) and resembled the joints of mice with simulation injection

or without injection (not shown). The quantification of joint inflammation revealed that although there was little exudation (cell mass in the joint cavity) in wild-type or non-tenascin C mice, infiltrate levels (cell mass in the synovial layer) were significantly reduced in mice without tenascin C (Figure 1f). Erosion of cartilage or bone did not occur in mice of any genotype (not shown), however there was a low level of chondrocyte death in wild-type mice, which was not observed in mice without tenascin C (Figure 1g) . Therefore the expression of tenascin C seems to promote the maintenance of acute inflammation.

Mice without tenascin C are protected from persistent inflammation and structural damage during antigen-induced arthritis

To determine whether tenascin C also contributes to more destructive joint inflammatory disease, erosive arthritis was induced by intraarticular injection of mBSA into the knee joint after immunization with mBSA. This model involves both cellular and humoral immune responses and induces pathological changes similar to human RA (Brackertz (1977)). Injection of mBSA induced a similar inflammatory response in mice without both tenascin C and wild type. Cell infiltration and synovial thickening is evident within 24 hours in mice of both genotypes (Figure 2c-f, h, i) compared to mice with simulation injection (Figure 2a, b, g) or not injected (not shown) .

However, this does not persist in mice without tenascin C as it does in wild-type mice. At 3 days after injection, wild-type mice show increased inflammation of the meniscus and capsule, synovial hyperplasia, cells and fibrin deposits in the joint space, pannus formation and loss of localized cartilage proteoglycans (Figure 3a , b, f). On the contrary, at 3 days in mice without tenascin C the inflammation is limited to the capsule, the synovial inflammation has decreased and there are no fibrin / cell aggregates present in the joint space, there is no pannus formation and there is no loss of cartilage proteoglycans (Figure 3c, d, e).

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At 7 days, wild-type mice showed persistent inflammatory cellular infiltration of the joint space, extensive synovitis and pannus formation and destruction of articular cartilage and bone erosion (Figure 4a, b). The knees to which simulation was injected and knees of mice that had not been injected were healthy and showed no inflammation or destruction of the joint (not shown). Tenascin C-deficient mice also had healthy joints that showed only mild inflammatory cell infiltration, without exudate from the joint space, synovitis, pannus formation, destruction of joint cartilage or bone erosion (Figure 4c, d). The joints of tenascin C-deficient mice that had been simulated and / or had not been injected were also healthy (not shown).

These histological data are reflected in the joint disease score as described in the materials and methods. The levels of cellular infiltrate and exudate observed in both wild-type mice and in mice without tenascin C 24 hours after injection were not significantly different. However, although cell mass continues to increase in wild-type mice over time, this response was attenuated in mice without tenascin C and the numbers of cells in the joint decreased over time (Figure 4e). Increasingly high levels of chondrocyte death appeared in the cartilage of wild-type mice over time, but no significant disease was observed in mice without tenascin C (Figure 4f). No superficial erosion of cartilage or bone erosion was apparent in wild-type mice at 24 hours or 3 days (not shown) but significant tissue destruction had occurred at 7 days. In contrast, mice without tenascin C showed no tissue destruction at 24 hours, 3 days (not shown) or 7 days (Figure 4f). These data indicate that although the onset of joint inflammation (inflow of cells into the synovium and joint space) is not affected in mice without tenascin C, unlike wild-type mice the disease does not progress until tissue destruction and cell death. These results demonstrate that tenascin C expression is required for persistent synovial inflammation and joint destruction in this model.

Example 4 - cell culture

Patient test samples-

Human monocytes were isolated from peripheral blood (London Blood Bank) and macrophages were derived from monocytes after differentiation for 4 days with 100 ng / ml of M-CSF as previously described (Foxwell (1998)).

Membrane cells were isolated with RA (representing a mixed population of all synovial cell types) from synovial membranes obtained from patients who underwent joint replacement surgery as previously described (Brennan (1989)). Synovial fibroblasts with RA were isolated from the mixed population of membrane cells with RA as previously described (Brennan (1989)). The study was approved by the ethics committee of the local foundation (Riverside NHS Research Committee) and residual tissue (synovium after joint replacement surgery) was obtained only after receiving signed informed consent from the patient and anonymizing the tissue to protect the patient identity

Immediately after isolation, membrane cells with RA and macrophages were cultured at 1 x 105 cells / well in RPMI 1640 containing 10% FBS (v / v) and 100 U / ml penicillin / streptomycin (Units / ml) in plates 96-well tissue culture for 24 hours before stimulation. Synovial fibroblasts (used only in pass number 2 or 3) were grown at 1 x 104 cells / well in DMEM containing 10% FBS (v / v) and 100 U / ml penicillin / streptomycin in 96 tissue culture plates wells for 24 hours before stimulation.

Mouse embryonic fibroblasts (MEF) and bone marrow derived macrophages (BMDM)

MEFs express high levels of mRNA from the 9 murine TLRs and are specifically and highly sensitive to TLR ligand activation. MEFs of mice with targeted deletions of TLR2, TLR4 and MyD88 demonstrate profound defects in their IL-6 response to specific ligands (Kurt-Jones (2004)). MEF were isolated from d13 embryos collected from mice of the same age, pregnant, wild-type, TLR2, TLR4 and null mice (as described in Todaro (1963)). Fibroblasts were cultured at 2 x 104 cells / well in DMEM containing 10% (v / v) FBS and 100 U / ml penicillin / streptomycin in 96-well tissue culture plates for 24 hours before stimulation.

BMDM were derived by aspirating the femurs of female wild-type mice of the same age, without TLR2 and TLR4 as described in Butler (1999)) and culturing the cells for 7 days in DMEM, 20% FBS (v / v), solution antibiotic-antifungal PSA 10 ml / l (v / v), -Mercaptoethanol 50 M and M-CSF 10 ng / ml. The macrophages were then cultured at 1 x 105 cells / well in DMEM, 20% FBS (v / v), 10 ml / l PSA antibiotic-antifungal solution, 50 M Merca-Mercaptoethanol in tissue culture plates 96 wells for 24 hours before stimulation.

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HEK293 cell lines

HEK293 cell lines expressing TLR2 and TLR4 / CD14 / MD-2 were cultured at 1 x 104 cells / well in DMEM containing 10% FBS (v / v) and 10 ug / ml blasticidine in 96-well tissue culture plates for 24 hours before stimulation.

Cellular stimulation and evaluation of cytokine synthesis

The cells were incubated for 24 hours at 37 ° C with the indicated doses of tenascin C and recombinant tenascin C fragments (1.0 M -1.0 nM). The cells were also stimulated when indicated with LPS (1 ng / ml for human macrophages, 10 ng / ml for human fibroblasts, membrane cells with RA and HEK, 100 ng / ml for MEFS and BMDM and 10 ng / ml for HEKS ), PAM3 (10 ng / ml for human macrophages, human fibroblasts and HEK, 100 ng / ml for MEF and BMDM), murine IL-1 (5 ng / ml for MEFS) and murine TNF- (100 ng / ml for MEFS). Unless specifically indicated otherwise, rough LPS was used for in vitro studies.

For adenoviral gene transfer experiments, synovial fibroblasts were incubated with human RA with adenoviral vectors at a multiplicity of infection of 100, washed after 2 hours, cultured in complete medium for 24 hours, then stimulated for 24 hours, after which were collected supernatants.

When indicated, cells were pre-incubated with 10 g / ml anti CD14 antibody, 10 g / ml IL1 receptor antagonist, 10 g / ml anti TLR2 antibody, 25 /g / ml anti TLR4 antibody, control antibody of isotype 10 or 25 /g / ml, polymyxin B 25 g / ml or msbB LPS 1 g / ml, for 30 minutes at 37 ° C before stimulation. Where indicated, recombinant tenascin C and FBG, and LPS were boiled for 15 minutes before addition to the cells.

In all cases, the viability of the cells was not significantly affected during the experimental time period when examined by the MTT cell viability test (Sigma, Poole, United Kingdom).

Supernatants were subsequently examined for the presence of TNF-, IL-6 and IL-8 cytokines by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. The absorbance was read on a spectrophotometric ELISA plate reader (Labsystems Multiscan Biochromic, Vantaa, Finland) and analyzed using the Ascent software program (Thermo Labsystems, Altrincham, United Kingdom).

Results

Tenascin C induces synthesis of TNF-, IL-6 and IL-8 in synovial fibroblasts with primary human RA and macrophages

The inventors then investigated whether tenascin C could activate the innate immune response. Tenascin C was used to stimulate primary human macrophages and synovial fibroblasts with RA and the production of the proinflammatory cytokines TNF-, IL-6 and IL-8 examined. The bacterial cell wall component LPS was used as a positive control. Tenascin C induced a specific cell-type cytokine profile that was significantly different from LPS. It stimulated dose-dependent production of TNF-, IL-6 and IL-8 in human macrophages (Figure 5a). However, tenascin C only induced synthesis of IL-6 in synovial fibroblasts, while LPS induced both IL-6 and IL-8 (Figure 5b). Neither LPS nor tenascin C induced synthesis of TNF- in fibroblasts (data not shown). Tenascin C stimulation of IL-6 (Figure 5c), IL-8 and TNF- by human macrophages and IL-6 by synovial fibroblasts (not shown) was heat sensitive and was not affected by the LPS inhibitor, polymyxin B. Together these results provide strong evidence that the induction of cytokines by tenascin C is not due to contamination with LPS.

The fibrinogen-type globule (FBG) mediates activation by tenascin C cells.

Tenascin C is a large hexamerican molecule, from which each domain binds to different cell surface receptors (reviewed in Orend (2005)). Understanding the mechanism of action of tenascin C will require the identification of which domain or domains are critical to promote cytokine production. The inventors synthesized recombinant proteins that comprised different domains of the molecule (Figure 10). Each domain was conducted in E. coli was purified (figure 11) and found to contain &lt;10 pg / ml of LPS by subjecting the pure protein to the Limulus amaebocyte lysate assay. Only one domain of tenascin C was active. The fibrinogen type globule (FBG) stimulated the synthesis of TNF- in human macrophages (Figure 6a), synthesis of IL-6 and IL-8 in human macrophages (not shown) and IL-6 in synovial fibroblasts with RA (not shown) to a degree equal to tenascin C of full length. As a full-length tenascin C, FBG did not induce synthesis of IL-8 in synovial fibroblasts with RA where LPS did (data not shown). FBG-induced cytokine synthesis was also sensitive to heat and was not affected by polymyxin B (data not shown).

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The tenascin C FBG domain induces the production of cytokines in the synovium with human RA and joint inflammation in mice.

The inventors investigated whether FBG could promote the expression of inflammatory cytokines in synovial membranes of patients with RA. This tissue model of RA (which included a mixed population of all synovial cell types) spontaneously produces high levels of IL-6, IL-8 and TNF- (Brennan (1989)) (Figure 6b). FBG further enhanced the synthesis of all these cytokines (Figure 6b). To determine whether FBG could induce inflammation in vivo, wild-type mice were injected intra-articularly FBG. The inventors observed a transient and dose-dependent stimulation of joint inflammation. There was no inflammation or loss of proteoglycans in non-injected mice or in mice injected with PBS (Figure 6c-e) or 100 ng of FBG (data not shown). In mice that were injected with 1 µg of FBG inflammatory cell infiltration (Figure 6f), mild synovitis, pannus formation (Figure 6g) and loss of proteoglycans (Figure 6h) were observed. A similar response was seen in mice that were injected with 3 µg of FBG (data not shown). After histological quantification, high levels of cell infiltrate and exudate and chondrocyte death were observed in mice injected with FBG, along with a modest amount of cartilage surface erosion and bone damage (Figure 6i).

FBG-mediated cytokine synthesis depends on Myd88

It has been shown that many DAMPs, including fibrinogen (Smiley (2001)), stimulate the innate immune response by TLR activation. Therefore, the inventors investigated whether TLRs could also mediate cytokine production induced by tenascin C. Myeloid differentiation factor 88 (MyD88) is required for signaling by all TLRs, except TLR3 (O'Neill (2008 )). Infection of synovial fibroblasts with adenovirus expressing dominant negative MyD88, but not GFP control virus, canceled the induction by FBG of IL-6 (Figure 7a). These data suggest that FBG-induced inflammation depends on functional MyD88. This effect of FBG does not appear to be mediated by IL-1 since the addition of the IL-1 receptor antagonist did not inhibit the induction of cytokines (data not shown). To confirm that the action of FBG is dependent on MyD88, the inventors demonstrated that FBG does not stimulate cytokine synthesis in embryonic fibroblasts isolated from mice with targeted deletions in the MyD88 gene. The TLR2 PAM3 ligand, TLR4 LPS and IL-1 ligand all signal by MyD88. Stimulation with these was also canceled in MEF of deficient mice. However, TNF-, which does not signal using MyD88, was not affected (Figure 7b). The retransfection of wild-type MyD88 restored the sensitivity of these cells to FBG, PAM3, LPS and IL-1 (data not shown).

FBG signals through TLR4

TLRs show specificity for endogenous ligands; Proteins are recognized by one or both of TLR2 and 4 (reviewed in O'Neill (2008)). Neutralizing antibodies for TLR4 inhibited the synthesis of IL-6, IL-8 and TNF- induced by both FBG and LPS in human macrophages and the synthesis of IL-6 in synovial fibroblasts with RA but had no effect on function of the TLR2 ligand, PAM3. Antibodies to TLR2 inhibited PAM3-mediated cytokine synthesis but had no effect on LPS-induced cytokine synthesis.

or FBG. Controls of the same isotype had no effect on cytokine synthesis induced by any ligand (TNF-síntesis synthesis by human macrophages is shown in Figure 8a). To confirm that the action of FBG is dependent on TLR4, the inventors demonstrated that FBG does not stimulate cytokine synthesis in embryonic fibroblasts or macrophages isolated from mice with targeted deletions in the TLR4 gene. FBG-mediated cytokine synthesis was not affected in embryonic fibroblasts or macrophages isolated from mice with targeted deletions in the TLR2 gene. Cells isolated from TLR2-deficient mice were not sensitive to PAM3, but were sensitive to LPS and IL-1. Cells isolated from TLR4-deficient mice were not sensitive to LPS but responded to OAM3 and IL-1 (Figure 8b, c). In addition, the expression of TLR4 was required for the arthritogenic action of FBG in vivo; FBG was able to induce joint inflammation in mice without TLR2 but not in mice without TLR4 (Figure 12).

Different co-receiver requirements for FBG and LPS

LPS signaling by TLR4 is mediated by a receptor complex that includes soluble MD-2 protein and soluble GPI or CD14 bound cell surface (reviewed in Fitzgerald (2004)). The inventors then examined whether CD14 and MD-2 are required for FBG activation of TLR4. As a positive control the inventors examined here the activity of smooth glycosylated LPS that requires both MD-2 and CD14 (Jiang (2005)). The synthesis of IL-6, IL-8 and TNF- mediated by LPS by human macrophages and the synthesis of IL-6 by synovial fibroblasts with AR was inhibited by anti-CD14 antibodies and an antagonistic LPS derived from the mutant E. coli msbB which competes for the union of LPS with MD-2 (Coats (2007)). In contrast, cytokine synthesis mediated both by PAM3, which does not require these co-receptors for TLR2 activation, or by FBG was not affected by anti-CD14 or mutant LPS antibodies to msbB (Figure 8d shows synthesis of TNF- by human macrophages). These data suggest that neither CD14 nor MD-2 are required for FBG-mediated cytokine synthesis. Therefore, although LPS and FBG signal both by activating TLR4, they may have different co-receptor requirements.

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Example 5 -inhibition of the action and synthesis of tenascin C in human tissue

This example studies the effect of (1) the prevention of the proinflammatory action of tenascin C and (2) the inhibition of the expression of tenascin C in the synovium with human RA. 5

Methods

Peptide synthesis

10 Nine overlapping peptides comprising the complete FBG domain (table 2) were synthesized by Biogenes, Germany. The peptides were cleaved at room temperature (cleavage mixture: 90% trifluoroacetate, 5% thioanisole, 3% ethanedithiol, 2% anisole), purified by reverse phase high performance liquid chromatography and characterized by MALDI mass spectroscopy analysis TOF The purity of the peptides was> 85% as determined by high performance liquid chromatography.

fifteen The team was unable to synthesize peptide 7, supposedly due to the formation of secondary structure that prevented elongation of the peptide chain (as previously indicated (LaFleur (1997))).

Table 2. Overlapping peptides spanning the complete FBG domain of human tenascin C

<dl><dt>Peptide number </dt><dd>Amino acid sequence </dd></dl>

<dl><dt>1 </dt><dd>TIGLLYPFPKDCSQAMLNGDTTSGLYTIYL </dd></dl>

<dl><dt>2 </dt><dd>YTIYLNGDKAEALEVFCDMTSDGGGWIVFL </dd></dl>

<dl><dt>3 </dt><dd>WIVFLRRKNGRENFYQNWKAYAAGFGDRRE </dd></dl>

<dl><dt>4 </dt><dd>GDRREEFWLGLDNLNKITAQGQYELRVD </dd></dl>

<dl><dt>5 </dt><dd>ELRVDLRDHGETAFAVYDKFSVGDAKTRYK </dd></dl>

<dl><dt>6 </dt><dd>KTRYKLKVEGYSGTAGDSMAYHNGRSFST </dd></dl>

<dl><dt>7 </dt><dd>RSFSTFDKDTDSAITNCALSYKGAFWYRN </dd></dl>

<dl><dt>8 </dt><dd>WYRNCHRVNLMGRYGDNNHSQGVNWFHWKG </dd></dl>

<dl><dt>9 </dt><dd>FHWKGHEHSIQFAEMKLRPSNFRNLEGRRKRA </dd></dl>

20

Patient test samples and cell culture

Membrane cells were isolated with RA (representing a mixed population of all synovial cell types) from synovial membranes obtained from patients undergoing replacement surgery.

25 articulation (Brennan (1989)). Synovial membrane tissue was digested in RPMI 1640 (GIBCO) containing 5% fetal calf serum (FCS) (GIBCO), collagenase type IV 5 mg / ml (Sigma) and DNAse type I 0.15 mg / ml (Sigma) and incubated at 37 ° C for 2 hours.

After incubation, the tissue was pipetted through a nylon mesh into a sterile beaker. The

30 cells were then washed three times in complete medium (RPMI 1640 supplemented with 10% FCS). Synovial fibroblasts with RA were isolated from the mixed population of membrane cells with RA by selection in DMEM (Bio-Whittaker) supplemented with 10% FBS, 1 µM glutamine, 100 U / ml penicillin and streptomycin. Human monocytes were isolated from peripheral blood (London Blood Bank) and monocyte macrophages were derived after differentiation for 4 days with 100 ng / ml of M-CSF.

35 The study was approved by the ethics committee of the local foundation, and residual tissue (synovium after joint replacement surgery) was obtained only after receiving signed informed consent from the patient and anonymizing the tissue to protect the patient's identity.

40 Cellular stimulation and evaluation of cytokine synthesis

Immediately after isolation, membrane cells were cultured with RA at 1 x 105 cells / well in RPMI 1640 containing 10% FBS (v / v) and 100 U / ml penicillin / streptomycin in 96-well tissue culture plates. Cells were incubated for 24 hours at 37 ° C without addition, with control buffer (PBS, 1% BSA, NaN3

Four. Five 0.01%) or with 25 M, 100 M or 250 M of each peptide encompassing FBG.

Synovial fibroblasts (used only in pass number 2 or 3) were seeded at a concentration of 5 x 104 cells in a 3.5 cm plate. SiRNA was transfected at a final concentration of 10 nM using Lipofectamine 2000

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(Invitrogen) for 4 hours in OptiMEM I without serum. Two different siRNAs against human tenascin C (s7069 and s229491) (Applied Biosystems) were used.

The s7069 siRNA sequences are: (sense 5 'CGCGAGAACUUCUACCAAAtt 3', antisense 5 'UUUGGUAGAAGUUCUCGCGtc 3') and s229491 are (5 'GGAAUAUGAAUAAAGAAGAtt 3', antisense 5 '3 UCUUAUUCUUG'. The siRNA against luciferase (Dharmacon) was transfected as an unguided control.

Four hours after transfection, the medium was changed with pre-equilibrated Dulbecco modified Eagle medium containing 10% (v / v) FBS and the cells were incubated for an additional 48 and 72 hours. The cells were then stimulated with 10 ng / ml LPS for 24 hours at 37 ° C. Tenascin C mRNA and protein levels were quantified by PCR and western blotting respectively. Total RNA was extracted from cells using a mini QiaAmp RNA Blood kit (Qiagen, Germany). CDNA was synthesized from equivalent amounts of total RNA using SuperScript® III Reverse Transcriptase (Invitrogen) and 18-unit oligo dT (Eurofins MWG Operon).

Gene expression was analyzed by ct delta-delta methods based on quantitative real-time PCR with the TaqMan primer set of human tenascin C (Hs01115663-m1) and endogenous control of human ribosomal protein (RPLPO) (4310879E) (Applied Biosystems ) on a Corbett Rotor-gene 6000 machine (Corbett Research Ltd). Tenascin C protein was detected in cell supernatants and cell lysates by SDS PAGE and western blotting using the MAB1908 antibody (Millipore).

Macrophages were grown at 1 x 105 cells / well in RPMI 1640 containing 5% (v / v) FBS and 100 U / ml penicillin / streptomycin in 96-well tissue culture plates for 24 hours before stimulation. Cells were incubated for 24 hours at 37 ° C without addition, with 1.0 M FNG, 1 ng / ml LPS or 1 or 20 FM FBG peptide. Where indicated, cells were pre-incubated with 20 µM FBG peptides for 15 minutes.

The viability of the cells was not significantly affected over the experimental period of time when examined by the MTT cell viability test (Sigma, Poole, United Kingdom). Supernatants were examined for the presence of TNF-, IL-6 and IL-8 cytokines by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (R&amp;D systems). The absorbance was read on a spectrophotometric ELISA plate reader (Labsystems Multiscan Biochromic, Vantaa, Finland) and analyzed using the Ascent software program (Thermo Labsystems, Altrincham, United Kingdom).

Statistical methods

The mean, DT and ETM were calculated using GraphPad (GraphPad Software Inc., San Diego, CA).

Results

Blocking the synthesis of cytokines in membrane cultures with RA by specific FBG peptides

The peptide inhibition approach has been used successfully to determine the inav3 integrin binding site in the tenascin C FBG domain and to prevent cell adhesion in response to this tenascin C domain (Lafleur (1997) and Yokoyama (2000)).

The inventors synthesized a series of 8 overlapping peptides of approximately 30 amino acids that encompassed the complete FBG sequence (Table 2). Peptides were tested for the ability to block the synthesis of spontaneous cytokines in synovial membrane cultures with RA. The synthesis of TNF and IL8 was inhibited by peptides 3 and 8, but not by any other peptide (TNF shown in Figure 15). Peptides 3 and 8 dose-dependently inhibited cytokine synthesis achieving the highest concentrations 95% and 56% inhibition respectively (Figure 16). Although peptide 5 had no effect on TNF synthesis, it blocked dose-dependent synthesis of IL8 in membrane cells with RA with a maximum inhibition of 81% (Figure 16).

To map the active domain within FBG responsible for inducing cytokine production, the inventors stimulated primary human macrophages with each FBG peptide. Peptides 1, 5 and 6 all induced cytokine synthesis in a dose-dependent manner (Figure 17).

To determine if any peptide could block FBG-induced cytokine synthesis in human macrophages, cells were pre-incubated with each FBG peptide before stimulation with complete FBG or LPS. Peptide 5 specifically blocked the synthesis of FBG-mediated cytokines, while peptide 8 blocked the synthesis of cytokines in response to both LPS and FBG (Figure 18).

Peptide 8 therefore non-specifically blocks the production of cytokines induced by any stimulus. This domain is the FBG integrin binding domain that mediates cell adhesion and therefore can act

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to prevent the union of cells with tissue culture plates. Peptide 5 specifically blocks FBG-induced cytokine synthesis suggesting that the direction of this domain may be useful in preventing tenascin C-induced inflammation.

Silencing the gene expression of tenascin C inhibits the synthesis of cytokines in synovial fibroblasts with RA

Examination of the effect of inhibition of tenascin C expression on synovium with human RA has identified synovial fibroblasts as the main source of tenascin C in RA (Figure 1 C) (in Goh 2010).

The siRNA-mediated cancellation of tenascin C expression in these cells has been shown with a maximum efficacy of 94-96% (Figure 19). In cells transfected with tenascin C siRNA, both the basal level of cytokine synthesis and the production of cytokines induced by LPS were inhibited in 38% and 44% respectively compared to control cells (Figure 19).

These data reveal that the silencing of tenascin C in synovial fibroblasts with RA reduces the synthesis of proinflammatory cytokines and suggests that the cancellation of tenascin C expression is a viable strategy to inhibit inflammation in the synovium.

This work has established that blocking the activity of tenascin C (with peptides) and the expression of tenascin C (with siRNA) reduces the synthesis of inflammatory cytokines in synoviums with human RA. These data show that tenascin C blockade is potentially clinically beneficial in the treatment of RA and other inflammatory diseases.

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